Method for preparing flour doughs and products made from such doughs using glycerol oxidase and lipase

ABSTRACT

A method of improving the rheological properties of a flour dough and the quality of bread, alimentary paste products, noodles and cakes produced therefrom, wherein a combination of (a) a glycerol oxidase which does not require a co-factor to oxidize glycerol, and (b) a lipase, is added to the dough to produce a synergistic effect upon said rheological properties; and dough improving compositions containing such components.

This application is a 371 of PCT/DK98/00136, filed Apr. 3, 1998.

FIELD OF THE INVENTION

The present invention relates to the field of food manufacturing, inparticular to the preparation of improved bakery products and otherfarinaceous food products. Specifically, the invention concerns the useof glycerol oxidase as a dough strengthening agent and improvement ofthe quality of baked and dried products made from such improved doughs.There is also provided a method of improving the properties of doughsand baked product by combined use of glycerol oxidase and a lipase.

TECHNICAL BACKGROUND AND PRIOR ART

The “strength” or “weakness” of doughs is an important aspect of makingfarinaceous finished products from doughs, including baking. The“strength” or “weakness” of a dough is primarily determined by itscontent of protein and in particular the content and the quality of thegluten protein is an important factor in that respect. Flours with a lowprotein content are generally characterized as “weak”. Thus, thecohesive, extensible, rubbery mass which is formed by mixing water andweak flour will usually be highly extensible when subjected to stress,but it will not return to its original dimensions when the stress isremoved.

Flours with a high protein content are generally characterized as“strong” flours and the mass formed by mixing such a flour and waterwill be less extensible than the mass formed from a weak flour, andstress which is applied during mixing will be restored without breakdownto a greater extent than is the case with a dough mass formed from aweak flour. Strong flour is generally preferred in most baking contextsbecause of the superior rheological and handling properties of the doughand the superior form and texture qualities of the finished baked ordried products made from the strong flour dough. Doughs made from strongflours are generally more stable. Stability of a dough is one of themost important characteristics of flour doughs. Within the bakery andmilling industries it is known to use dough “conditioners” to strengthenthe dough to increase its stability and strength. Such doughconditioners are normally non-specific oxidizing agents such as e.g.iodates, peroxides, ascorbic acid, K-bromate or azodicarbonamide andthey are added to dough with the aims of improving the bakingperformance of flour to achieve a dough with improved stretchability andthus having a desirable strength and stability. The mechanism behindthis effect of oxidizing agents is that the flour proteins, inparticular gluten contains thiol groups which, when they becomeoxidized, form disulphide bonds whereby the protein forms a more stablematrix resulting in a better dough quality and improvements of thevolume and crumb structure of the baked products.

However, the use of several of the currently available non-specificoxidizing agents is either objected to by consumers or is not permittedby regulatory bodies. Hence it has been attempted to find alternativesto these conventional flour and dough additives, and the prior art hasi.a. suggested the use of glucose oxidase and hexose oxidase for thispurpose.

Glycerol oxidase is an oxidoreductase which is capable of oxidizingglycerol. Different types of glycerol oxidase have been described in theliterature. Some of these glycerol oxidases need co-factors in order tooxidize glycerol (Shuen-Fu et al., 1996. Enzyme Micro. Technol.,18:383-387).

However, glycerol oxidase from Aspergillus japonicus does not requireany co-factors in the oxidation of glycerol to glyceraldehyd (T. Uwajimaand O. Terada, 1980. Agri. Biol. Chem. 44:2039-2045).

This glycerol oxidase has been characterized by T. Uwajima and O. Terada(Methods in Enzymology, 1982, 89:243-248) and T. Uwajima et al. (Agric.Biol. Chem., 1979, 43:2633-2634), and has a pH optimum at 7.0 and K_(m)and V_(max) are 10.4 mM and 935.6 μmol H₂O₂ min⁻¹ respectively usingglycerol as substrate. The enzyme is most active on glycerol but alsoother substrates like dihydroxyacetone, 1,3-propanediol, D-galactose adD-fructose are oxidized by glycerol oxidase.

Glycerol oxidase not requiring co-factors has also been isolated fromPenicillium and characterized by Shuen-Fuh Lin et al. (Enzyme Micro.Technol., 1996, 18:383-387). This enzyme has optimum activity in the pHrange from 5.5 to 6.5 at 30° C. The enzyme is stable between 20 and 40°C. but loses its activity at temperatures above 50° C.

Other potential sources for glycerol oxidase according to the inventioninclude different fungal species as disclosed in DE-2817087-A, such asAspergillus oryzae, Aspergillus parasiticus, Aspergillus flavus,Neurospora crassa, Neurospora sitophila, Neurospora tetrasperma,Penicillium nigricans, Penicillium funiculosum and Penicilliumjanthinellum.

Glycerol oxidase isolated from the above natural sources has been usedfor different applications. Thus, glycerol oxidase from Aspergillusjaponicus has been used for glycoaldehyde production from ethyleneglycol (Kimiyasu Isobe and Hiroshi Nishise, 1995, Journal of MolecularCatalysis B: Enzymatic, 1:37-43). Glycerol oxidase has also been used inthe combination with lipoprotein lipase for the determination ofcontaminated yolk in egg white (Yioshinori Mie, 1996. Food ResearchInternational, 29:81-84). DE-2817087-A and U.S. Pat. No. 4,399,218disclose the use of glycerol oxidase for the determination of glycerol.

It has now been found that the addition of a glycerol oxidase to a flourdough results in an increased resistance hereof to deformation when thedough is stretched, i.e. this enzyme confers to the dough an increasedstrength whereby the dough becomes less prone to mechanical deformation.Accordingly, glycerol oxidase is highly useful as a dough conditioningagent in the manufacturing of flour dough based products including notonly bread products but also other products made from flour doughs suchas noodles and alimentary paste products.

It has also been found that the dough strengthening effect of glyceroloxidase is potentiated significantly when it is combined with a lipase,which in itself does not affect the dough strength. Furthermore, thecombined use of glycerol oxidase and lipase results in an improvement ofbread quality, in particular in respect of specific volume and crumbhomogeneity, which is not a simple additive effect, but reflects asynergistic effect of these two types of enzymes.

SUMMARY OF THE INVENTION

Accordingly, the invention relates in a first aspect to a method ofimproving the rheological properties of a flour dough and the quality ofthe finished product made from the dough, comprising adding to the dough10 to 10,000 units of a glycerol oxidase per kg of flour.

In a further aspect there is provided a method of improving therheological properties of a flour dough and the quality of the finishedproduct made from the dough, comprising adding to the dough a glyceroloxidase and a lipase.

The invention pertains in a still further aspect to dough improvingcomposition comprising a glycerol oxidase and at least one further doughingredient or dough additive.

In still further aspects, the invention relates to the use of a glyceroloxidase for improving the rheological properties of a flour dough andthe quality of the finished product made from the dough and to the useof a glycerol oxidase and a lipase in combination for improving therheological properties of a flour dough and the quality of the finishedproduct made from the dough.

DETAILED DISCLOSURE OF THE INVENTION

In one aspect, the present method provides a method of improving therheological properties of flour doughs.

The expression “rheological properties” as used herein refersparticularly to the effects of dough conditioners on dough strength andstability as the most important characteristics of flour doughs.According to American Association of Cereal Chemists (AACC) Method36-01A the term “stability” can be defined as “the range of dough timeover which a positive response is obtained and that property of arounded dough by which it resists flattening under its own weight over acourse of time”. According to the same method, the term “response” isdefined as “the reaction of dough to a known and specific stimulus,substance or set of conditions, usually determined by baking it incomparison with a control”

As it is mentioned above, it is generally desirable to improve thebaking performance of flour to achieve a dough with improvedstretchability and thus having a desirable strength and stability byadding oxidizing agents which cause the formation of protein disulphidebonds whereby the protein forms a more stable matrix resulting in abetter dough quality and improvements of the volume and crumb structureof baked products.

Thus, the term “rheological properties” relates to the above physicaland chemical phenomena which in combination will determine theperformance of flour doughs and thereby also the quality of theresulting products.

The method comprises, as it is mentioned above, the addition of aneffective amount of a glycerol oxidase to the dough. It will beunderstood that the addition can be either to a component of the doughrecipe or to the dough resulting from mixing all of the components forthe dough. In the present context, “an effective amount” is used toindicate that the amount is sufficient to confer to the dough and/or thefinished product improved characteristics as defined herein.Specifically, such an amount is in the range of 10 to 10,000 units ofglycerol oxidase per kg flour.

In one useful embodiment of the method according to the invention, theglycerol oxidase can, as it is described in details herein, be isolatedfrom a bacterial species, a fungal species, a yeast species, an animalcell including a human cell or a plant cell. Examples of glyceroloxidase producing fungal species are species belonging to the generaAspergillus, Neurospora and Penicillium, such as A. japonicus, A.oryzae, A. parasiticus, A. flavus, Neurospora crassa, N. sitophila, N.tetrasperma, Penicillium nigricans, P. funiculosum and P. janthinellum.

Glycerol oxidase can be derived as a native enzyme from natural sourcessuch as the above.

It is one objective of the invention to provide improved bakeryproducts. In accordance with the invention, a bakery product doughincluding a bread dough is prepared by mixing flour with water, aleavening agent such as yeast or a conventional chemical leaveningagent, and an effective amount of glycerol oxidase under dough formingconditions. It is, however, within the scope of the invention thatfurther components can be added to the dough mixture.

Typically, such further dough components include conventionally useddough components such as salt, sweetening agents such as sugars, syrupsor artificial sweetening agents, lipid substances including shortening,margarine, butter or an animal or vegetable oil, glycerol and one ormore dough additives such as emulsifying agents, starch degradingenzymes, cellulose or hemicellulose degrading enzymes, proteases,lipases, non-specific oxidizing agents such as those mentioned above,flavouring agents, lactic acid bacterial cultures, vitamins, minerals,hydrocolloids such as alginates, carrageenans, pectins, vegetable gumsincluding e.g. guar gum and locust bean gum, and dietary fibersubstances.

Conventional emulsifying agents used in making flour dough productsinclude as examples monoglycerides, diacetyl tartaric acid esters ofmono- and diglycerides of fatty acids, and lecithins e.g. obtained fromsoya. Among starch degrading enzymes, amylases are particularly usefulas dough improving additives. Other useful starch degrading enzymeswhich may be added to a dough composition include glucoamylases andpullulanases. In the present context, further interesting enzymes arexylanases and oxidoreductases such as glucose oxidase, pyranose oxidase,hexose oxidase, sulfhydryl oxidase, and lipases.

A preferred flour is wheat flour, but doughs comprising flour derivedfrom other cereal species such as from rice, maize, barley, rye anddurra are also contemplated.

In accordance with the invention, the dough is prepared by admixingflour, water, the glycerol oxidase and optionally other ingredients andadditives. The glycerol oxidase can be added together with any doughingredient including the water or dough ingredient mixture or with anyadditive or additive mixture. The dough can be prepared by anyconventional dough preparation method common in the baking industry orin any other industry making flour dough based products.

The glycerol oxidase can be added as a liquid preparation or in the formof a dry powder composition either comprising the enzyme as the soleactive component or in admixture with one or more other doughingredients or additive.

The amount of the glycerol oxidase added is an amount which results inthe presence in the dough of 10 to 5,000 units (as defined in thefollowing) such as 10 to 2,500 units per kg of flour. In usefulembodiments, the amount is in the range of 20 to 1,500 units per kg offlour.

The effect of the glycerol oxidase on the Theological properties of thedough can be measured by standard methods according to the InternationalAssociation of Cereal Chemistry (ICC) and the American Association ofCereal Chemistry (AACC) including the amylograph method (ICC 126), thefarinograph method (AACC 54-21) and the extensigraph method (AACC54-10). The AACC method 54-10 defines the extensigraph in the followingmanner: “the extensigraph records a load-extension curve for a testpiece of dough until it breaks. Characteristics of load-extension curvesor extensigrams are used to assess general quality of flour and itsresponses to improving agents”. In effect, the extensigraph methodmeasures the relative strength of a dough. A strong dough exhibits ahigher and, in some cases, a longer extensigraph curve than does a weakdough.

In a preferred embodiment of the method according to the invention, theresistance to extension of the dough in terms of the ratio between theresistance to extension (height of curve, B) and the extensibility(length of curve, C), i.e. the B/C ratio as measured by the AACC method54-10 is increased by at least 10% relative to that of an otherwisesimilar dough not containing glycerol oxidase. In more preferredembodiments, the resistance to extension is increased by at least 20%,such as at least 50% and in particular by at least 100%.

It has been found that the addition of glycerol oxidase to bakeryproduct doughs results in bakery products such as yeast leavened andchemically leavened products in which the specific volume is increasedrelative to an otherwise similar bakery product, prepared from a doughnot containing glycerol oxidase. In this context, the expression“specific volume” is used to indicate the ratio between volume andweight of the product. It has been found that, in accordance with theabove method, the specific volume can be increased significantly such asby at least 10%, preferably by at least 20%, including by at least 30%,preferably by at least 40% and more preferably by at least 50%.

The method according to the invention is highly suitable for improvingthe rheological properties and quality of the finished products ofconventional types of yeast leavened bread products based on wheatflour, such as loaves and rolls. The method is also suitable forimproving the rheological properties of doughs containing chemicalleavening agents (baking powder) and the quality of products made fromsuch doughs. Such product include as examples sponge cakes and muffins.

In one interesting aspect, the invention is used to improve theTheological properties of doughs intended for noodle products including“white noodles” and “chinese noodles” and to improve the texturalqualities of the finished noodle products. A typical basic recipe forthe manufacturing of noodles comprises the following ingredients: wheatflour 100 parts, salt 0.5 parts and water 33 parts. Furthermore,glycerol is often added to the noodle dough. The noodles are typicallyprepared by mixing the ingredients in an appropriate mixing apparatusfollowed by rolling out the noodle dough using an appropriate noodlemachine to form the noodle strings which are subsequently air dried.

The quality of the finished noodles is assessed i.a. by their colour,cooking quality and texture. The noodles should cook as quickly aspossible, remain firm after cooking and should preferably not loose anysolids to the cooking water. On serving the noodles should preferablyhave a smooth and firm surface not showing stickiness and provide a firm“bite” and a good mouthfeel. Furthermore, it is important that the whitenoodles have a light colour.

Since the appropriateness of wheat flour for providing noodles havingthe desired textural and eating qualities may vary according to the yearand the growth area, it is usual to add noodle improvers to the dough inorder to compensate for sub-optimal quality of the flour. Typically,such improvers will comprise dietary fiber substances, vegetableproteins, emulsifiers and hydrocolloids such as e.g. alginates,carrageenans, pectins, vegetable gums including guar gum and locust beangum, and amylases, and as mentioned above, glycerol.

It is therefore an important aspect of the invention that the glyceroloxidase according to the invention is useful as a noodle improving agentoptionally in combination with glycerol and other components currentlyused to improve the quality of noodles. Thus, it is contemplated thatnoodles prepared in accordance with the above method will have improvedproperties with respect to colour, cooking and eating qualitiesincluding a firm, elastic and non-sticky texture and consistency.

In a further useful embodiment, the dough which is prepared by themethod according to the invention is a dough for preparing an alimentarypaste product. Such products which include as examples spaghetti andmaccaroni are typically prepared from a dough comprising mainingredients such as flour, eggs or egg powder and/or water. After mixingof the ingredient, the dough is formed to the desired type of pasteproduct and air dried. It is contemplated that the addition of glyceroloxidase to a paste dough, optionally in combination with glycerol, willhave a significant improving effect on the extensibility and stabilityhereof resulting in finished paste product having improved textural andeating qualities.

In a useful embodiment, there is provided a dough improving methodwherein at least one further enzyme is added to the dough ingredient,dough additive or the dough. In the present context, suitable enzymesinclude cellulases, hemicellulases, xylanases, starch degrading enzymes,oxidoreductases and proteases.

In a further aspect, the invention relates to a method of improving therheological properties of a flour dough and the quality of the finishedproducts made from the dough which comprises that both a glyceroloxidase and a lipase is added to the dough.

It was surprisingly found that the two types of enzymes were capable ofinteracting with each other under the dough conditions to an extentwhere the effect on improvement of the dough strength and bread qualityby the enzymes was not only additive, but the effect was synergistic.

Thus, with respect to improvement of dough strength it was found thatwith glycerol oxidase alone, the B/C ratio as measured after 45 minutesof resting was increased by 34%, with lipase alone no effect wasobserved. However, when combining the two enzymes, the B/C ratio wasincreased by 54%, i.e. combining the glycerol oxidase with the lipaseenhanced the dough strengthening effect of glycerol oxidase by more than50%. Thus, one objective of combining glycerol oxidase and a lipase isto provide an enhancement of the dough strengthening effect of glyceroloxidase by at least 25% such as at least 50% including at least 75%,determined as described herein.

In relation to improvement of finished product, it was found that thecombined addition of glycerol oxidase and a lipase resulted in asubstantial synergistic effect in respect to crumb homogeneity asdefined herein. Also, with respect to the specific volume of bakedproduct a synergistic effect was found. Thus, for a bread product, theaddition of lipase alone typically results in a negligible increase ofthe specific volume, addition of glycerol oxidase alone in an increaseof about 25%, whereas a combined addition of the two enzymes results inan increase of more than 30%.

Further in relation to improvement of the finished product, it was foundthat the addition of lipase resulted in modification of the glycolipids,monogalactosyl diglyceride and digalactosyl diglyceride present indough. These components were converted to the more polar componentsmonogalactosyl monoglyceride and digalactosyl monoglyceride. Asgalactosyl monoglycerides are more surface active components thangalactosyl diglycerides it is assumed that galactosyl monoglyceridescontributed to the observed improved crumb cell structure andhomogeneity. Thus, one objective of using lipase is to hydolyse at least10% of the galactosyl diglycerides normally present in a flour dough tothe corresponding galactosyl monoglycerides, such as at least 50%including at least 100%.

The details of such a method using combined addition of glycerol oxidaseand lipase are, apart from the use of a lipase in combination withglycerol oxidase, substantially similar to those described above for amethod according to the invention which does not require the addition ofa lipase.

When using, in accordance with the invention, a lipase in combinationwith a glycerol oxidase, the amount of lipase is typically in the rangeof 10 to 100,000 lipase units (LUS) (as defined in the following) per kgflour including the range of 10 to 20,000 LUS, e.g. 100 to 15,000 LUSsuch as 500 to 10,000 LUS.

Lipases that are useful in the present invention can be derived from abacterial species, a fungal species, a yeast species, an animal cell anda plant cell. Whereas the enzyme may be provided by cultivating culturesof such source organisms naturally producing lipase, it may be moreconvenient and cost-effective to produce it by means of geneticallymodified cells such as it is described in details in the followingexamples. In the latter case, the term “derived” may imply that a genecoding for the lipase is isolated from a source organism and insertedinto a host cell capable of expressing the gene.

Thus, the enzyme may in a useful embodiment be derived from anAspergillus species including as examples A. tubigensis, A. oryzae andA. niger.

Presently preferred lipases include the lipase designated Lipase 3, theproduction and characteristics of which is described in details in thefollowing examples, or a mutant of this enzyme. In the present context,the term “mutant” refers to a lipase having, relative to the wild-typeenzyme, an altered amino acid sequence. A further preferred lipase isthe lipase found in the commercial product, GRINDAMYL™ EXEL 16.

In a further aspect of the invention there is provided a dough improvingcomposition comprising a glycerol oxidase and at least one further doughingredient or dough additive.

The further ingredient or additive can be any of the ingredients oradditives which are described above. The composition may conveniently bea liquid preparation comprising the glycerol oxidase. However, thecomposition is conveniently in the form of a dry composition.

The amount of the glycerol oxidase in the composition is in the range of10 to 10,000 units per kg flour. It will be appreciated that thisindication of the amount of enzyme implies that a recommendedappropriate amount of the composition will result in the above statedamount in the dough to which it is added. In specific embodiments, theamount of glycerol oxidase is in the range of 10 to 5,000 units such as10 to 2,500 units per kg of flour. In other useful embodiments, theamount is in the range of 20 to 1,500 units per kg of flour.

In another embodiment, the dough improving composition may furthercomprises a lipase as defined above and in the amounts as also describedabove in relation to the method according to the invention.

Optionally, the composition is in the form of a complete dough additivemixture or pre-mixture for making a particular finished product andcontaining all of the dry ingredients and additives for such a dough. Inspecific embodiments, the composition is one particularly useful forpreparing a baking product or in the making of a noodle product or analimentary paste product.

In one advantageous embodiment of the above method at least one furtherenzyme is added to the dough. Suitable examples hereof include acellulase, a hemicellulase, a xylanase, a starch degrading enzyme,hexose oxidase and a protease.

In a preferred advantageous embodiment, the further added enzyme is alipase. It has been found that in accordance with the above method, thecrumb homogeneity and specific volume of the bakery product can beincreased significantly as compared to that of an otherwise similarbakery product prepared from a dough not containing glycerol oxidase,and from a similar bakery product prepared from a dough containingglycerol oxidase.

In a still further aspect, the present invention pertains to the use ofa glycerol oxidase and a lipase in combination for improving therheological properties of a flour dough and the quality of the finishedproduct made from the dough.

In this connection, specific embodiments include use wherein theimprovement of the rheological properties of the dough include that theresistance to extension of the dough in terms of the ratio betweenresistance to extension (height of curve, B) and the extensibility(length of curve, C), i.e. the B/C ratio, as measured by the AACC method54-10 is increased by at least 10% relative to that of an otherwisesimilar dough that does not contain glycerol oxidase and use wherein theimprovement of the quality of the finished product made from the doughis that the average pore diameter of the crumb of the bread made fromthe dough is reduced by at least 10%, relative to a bread which is madefrom a bread dough without addition of the lipase.

In a further embodiment, the use according to the invention, impliesthat the improvement of the quality of the finished product made fromthe dough consists in that the pore homogeneity of the crumb of thebread made from the dough is incresed by at least 5%, relative to abread which is made from a bread dough without addition of the lipase.The pore homogeneity of bread is conveniently measured by means of animage analyzer composed of a standard CCD-video camera, a videodigitiser and a personal computer with WinGrain software. Using such ananalyzer, the results of pore diameter in mm and pore homogeneity can becalculated as an average of measurements from 10 slices of bread. Thepore homogeneity is expressed in % of pores that are larger than 0.5times the average of pore diameter and smaller than 2 times the averagediameter.

In a further embodiment, the use relates to improvement of therheological characteristics of the dough including that the gluten index(as defined hereinbelow) in the dough is increased by at least 5%,relative to a dough without addition of a lipase, the gluten index isdetermined by means of a Glutomatic 2200 apparatus.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is further illustrated by reference to theaccompanying figures in which

FIG. 1 shows the restriction map of the genomic clone of the lipA gene,

FIG. 2 shows the structure of the lipA gene encoding lipase 3,

FIG. 3 shows a chromatogram of HIC fractionated culture supernatant ofan Aspergillus tubigensis transformant with 62-fold increase of lipase3, and

FIG. 4 shows a chromatogram of HIC fractionated culture supernatant ofthe untransformed Aspergillus tubigensis strain.

The invention will now be described by way of illustration in thefollowing non-limiting examples.

A. PRODUCTION AND PURIFICATION OF GLYCEROL OXIDASE (GLOX) EXAMPLE 1Production, extraction and purification of glycerol oxidase usingdifferent strains and cultivation conditions

1. Production, extraction and purification of glycerol oxidase usingAspergillus japonicus ATCC 1042 cultivated in a production mediumcontaining 3% glycerol

The following assay for determination of glycerol oxidase activity wasused:

The assay is based on the method described by Sullivan and Ikawa(Biochimica and Biophysica Acta, 1973, 309:11-22), but modified asdescribed in the following. An assay mixture containing 150 μl 2%glycerol (in 100 mM phophate buffer, pH 7.0), 120 μl 100 mM phosphatebuffer, pH 7.0, 10 μl o-dianisidin dihydrochloride (Sigma D 3252, 3mg/ml in H₂O), 10 μl peroxidase (POD) (Sigma P8125, 0.1 mg/ml in 100 mMphosphate buffer, pH 7.0) and 10 μl glycerol oxidase (GLOX) solution.The controls are made by adding buffer in place of GLOX solution. Theincubation is started by the addition of glycerol. After 15 minutes ofincubation at 25° C. in microtiter plates, the absorbance at 402 nm isread in a Elisa reader. A standard curve is constructed using varyingconcentrations of H₂O₂ in place of the enzyme solution. The reaction canbe described in the following manner:

Oxidised o-dianisidine has a yellow colour absorbing at 402 nm.

One glycerol oxidase unit (U) is the amount of enzyme which catalysesthe production of 1 μmole H₂O₂ per minute at 25° C., pH 7.0 at asubstrate concentration of 0.2 M glycerol.

A spore suspension of Aspergillus japonicus ATCC 1042 was prepared byincubating A. japonicus on PDA medium (30° C., 7 days) and washing with10 ml of 0.2% Tween 80. A preculture was prepared by inoculating 1 ml ofthe resulting spore suspension in 300 ml production medium containing3.0% of glycerol (87%, Merck), 0.3% of yeast extract (Difco), 0.1% ofmeat extract (Difco), 0.1% KH₂PO₄ (Merck), 0.1% of MGSO₄*7H₂O (Merck),0.1% antifoam (Contra spum) and 70 mg/l of chloramphenicolum(Mecobenzon) (pH adjusted to 7.2 with NaOH) in a 500 ml flask. Thepreculture was incubated overnight at 30° C. with shaking (200 rpm).

A 30 liter fermenter with 15 liter production medium was inoculated with900 ml (corresponding to 3 flasks) of the resulting overnightpreculture, and cultured at 30° C. for 25 hours under continuousstirring (350 rpm) and aeration (15 l/min). After culturing, the myceliawas harvested from the resulting culture broth by filtration on aWhatman GF/B filter by suction, and washed with 3 liters of deionizedwater. The mycelium yield was 186 g (wet weight).

A part (50 g) of the resulting mycelial mat was suspended in 700 ml of50 mM borate buffer (pH 10.0), and disrupted by ultrasonication(Branson, Sonifer 250) at 5° C. (3×5 minutes). After disruption, themycelia was removed by centrifugation (29,000 g for 15 minutes), thecell-free extract (700 ml) was brought to 40% saturation with ammoniumsulfate and the resulting precipitate was removed by centrifugation(29,000 g for 20 minutes). The ammonium sulfate concentration was thenincreased to 70% saturation to precipitate the enzyme. The resultingprecipitate was collected and solubilized in 100 ml of 50 mM boratebuffer (pH 10.0). The crude extract was then dialysed for 24 hoursagainst 5 l of 50 mM borate buffer (pH 10.0). After dialysis theinsoluble matters in the crude extract were removed by centrifugation(18,000×g for 10 minutes). The resulting supernatant contained 8.7 unitsof glycerol oxidase activity per ml.

2. Production extraction and purification of glycerol oxidase usingAspergillus japonicus ATCC 1042 cultivated in a production mediumcontaining 5% glycerol

A spore suspension of Aspergillus japonicus ATCC 1042 was prepared asdescribed above. A preculture was prepared by inoculating 1 ml of theresulting spore suspension into a flask (500 ml) containing 200 mlproduction medium (5.0% glycerol, 0.25% yeast extract, 0.1% Maltextract, 0.7% antifoam (Contra spum), pH adjusted to 6.2 with HCl,sterilization at 121° C. for 90 minutes). The preculture was incubated 3days at 30° C. with continuous shaking (200 rpm). A 6 liter fermenterwith 5 liter production medium as described above was inoculated with 50ml of the resulting preculture and cultured at 30° C. for 3 days undercontinuous stirring (250 rpm) and aeration (5 l/min). After culturingthe mycelia was harvested from the resulting culture broth by filtrationon a Whatman GF/B filter by suction, and washed with 3 liter ionizedwater containing 0.9% NaCl. The resulting mycelia mat was frozen inliquid nitrogen, suspended in 200 ml of 50 mM phosphate buffer (pH 7.0)and disrupted by ultrasonication (Branson, Sonifer 250) at 5° C. (4minutes). After disruption, the mycelia was removed by filtration on aWhatman GF/A filter by suction. The enzyme in the resulting filtrate wasconcentrated on a AMICON® 8400 ultrafiltration unit and contained 87units of glycerol oxidase per ml after ultrafiltration.

3. Production, extraction and purification of glycerol oxidase usingAspergillus japonicus ATCC 1042 cultivated in a production mediumcontaining 10% glycerol

A spore suspension of Aspergillus japonicus ATCC 1042 was prepared asdescribed above. A 1 ml sample of the resulting spore suspension wasinoculated into each of 5 flasks (500 ml) with 200 ml production mediumcontaining 10.0% of glycerol, 0.1% of yeast extract and 0.1% of maltextract (pH adjusted to 6.2 with HCl, sterilization at 121° C. for 15minutes). The cultures were incubated for 5 days at 30° C. with shaking(140 rpm).

The extraction and concentration of the enzyme was carried out asdescribed above. The resulting filtrate contained 66 units of glyceroloxidase per ml after ultrafiltration.

4. Production of glycerol oxidase from Penicillium funiculosum andPenicillium janthinellum

Spore suspensions of Penicillium funiculosum NRRL 1132 and Penicilliumjanthinellum NRRL 2016 were prepared as described above. A 1 ml sampleof each of the resulting spore suspensions was inoculated into separateflasks (1000 ml) containing 100 g wheat bran and 100 ml water (twoflasks for each culture)

Glycerol oxidase was extracted by suspending the wheat bran cultures in900 ml of 30 mM phosphate buffer (pH 6.5) containing 0.1% Triton X100(Merck). The mycelial mat was removed from the cultivation media byfiltration using a Whatman GF/B filter. The resulting mycelia mat wasfrozen in liquid nitrogen, suspended in 200 ml of 50 mM phosphate buffer(pH 7.0) and disrupted by ultrasonication (Branson, Sonifer 250) at 5°C. (4 minutes). After disruption, the mycelia was removed by filtrationon a Whatman GF/A filter by suction. The resulting filtrate from thePenicillium funiculosum culture contained 7.4 units of glycerol oxidaseper ml, and the resulting filtrate from the Penicillium janthinellumculture contained 11.3 units of glycerol oxidase per ml.

B. PRODUCTION, PURIFICATION AND CHARACTERIZATION OF Aspergillustubigensis LIPASE 3 MATERIALS AND METHODS

(i) Determination of lipase activity and protein

1. Plate assay on tributyrin-containing medium

The assay is modified from Kouker and Jaeger (Appl. Environ. Microbiol.,1987, 53:211-213).

A typical protocol for this assay is as follows: 100 ml 2% agar in 50 mMsodium phosphate buffer (pH 6.3) is heated to boiling, and after coolingto about 70° C. under stirring, 5 ml 0.2% Rhodamine B is added understirring plus 40 ml of tributyrin. The stirring is continued for 2minutes. The mixture is then sonicated for 1 minute. After an additional2 minutes of stirring, 20 ml of the agar mixture is poured intoindividual petri dishes. In the absence of lipase activity, the agarplates containing tributyrin and Rhodamine B will appear opaque and arepink coloured.

To quantify lipase activity, holes having a diameter of 3 mm are punchedin the above agar and filled with 10 μl of lipase preparation. Theplates are incubated for varying times at 37° C. When lipase activity ispresent in the applied preparation to be tested, a sharp pink/reddishzone is formed around the holes. When the plates are irradiated with UVlight at 350 nm, the lipase activity is observed as halos of orangecoloured fluorescence.

2. Modified Food Chemical Codex assay for lipase activity

Lipase activity based on hydrolysis of tributyrin is measured accordingto Food Chemical Codex, Forth Edition, National Academy Press, 1996, p.803. With the modification that the pH is 5.5 instead of 7. One LUT(lipase unit tributyrin) is defined as the amount of enzyme which canrelease 2 μmol butyric acid per min. under the above assay conditions.

3. p-nitrophenyl acetate assay

Lipase activity can also be determined colorimetrically usingp-nitrophenyl acetate as a substrate e.g. using the following protocol:In a microtiter plate 10 μl of sample or blank is added followed by theaddition of 250 μl substrate (0.5 mg p-nitrophenyl acetate per ml 50 mMphosphate buffer, pH 6.0). The microtiter plate is incubated for 5minutes at 30° C. and the absorbance at 405 nm is read using amicroplate reader. 1 unit is defined as 1 μmol p-nitrophenol releasedper 5 minutes.

4. p-nitrophenyl hexanoate assay

Lipase activity can be determined by using p-nitrophenyl hexanoate as asubstrate. This assay is carried out by adding 10 μl of samplepreparation or blank to a microtiter plate followed by the addition of250 μl substrate (0.5 mg p-nitrophenyl hexanoate per ml of 20 mMphosphate buffer, pH 6.). At this concentration of substrate thereaction mixture appears as a milky solution. The microtiter plate isincubated for 5 minutes at 30° C. and the absorbance at 405 nm is readin a microplate reader.

5. Titrimetric assay of lipase activity

Alternatively, lipase activity is determined according to Food ChemicalCodex (3rd Ed., 1981, pp 492-493) modified to sunflower oil and pH 5.5instead of olive oil and pH 6.5. The lipase activity is measured as LUS(lipase units sunflower) where 1 LUS is defined as the quantity ofenzyme which can release 1 μmol of fatty acids per minute from sunfloweroil under the above assay conditions.

6. Protein measurement

During the course of purification of lipase as described in thefollowing, the protein eluted from the columns was measured bydetermining absorbance at 280 nm. The protein in the pooled samples wasdetermined in microtiter plates by a sensitive Bradford method accordingto Bio-Rad (Bio-Rad Bulletin 1177 EG, 1984). Bovine serum albumin wasused as a standard.

EXAMPLE 2 Production, purification and characterization of lipase 3

2.1. Production

A mutant strain of Aspergillus tubigensis was selected and used for theproduction of wild type lipase. This lipase is referred to herein aslipase 3. The strain was subjected to a fermentation in a 750 lfermenter containing 410.0 kg of tap water, 10.8 kg soy flour, 11.1 kgammonium monohydrogenphosphate, 4.0 kg phosphoric acid (75%), 2.7 kgmagnesium sulfate, 10.8 kg sunflower oil and 1.7 kg antifoam 1510. Thesubstrate was heat treated at 121° C. for 45 minutes. The culture mediawas inoculated directly with 7.5×10⁹ spores of the mutant strain. Thestrain was cultivated for three days at 38° C., pH controlled at 6.5,aeration at 290 l/min and stirring at 180 rpm the first two days and at360 rpm the last day. The fermentate was separated using a drum filterand the culture filtrate was concentrated 3.8 times by ultra-filtration.The concentrated filtrate was preserved with potassium sorbate (0.1%)and sodium benzoate (0.2%) and used as a starting material forpurification of lipase.

2.2. Purification of lipase

A 60 ml sample of ferment (cf. 2.1) containing 557 LUS/ml, pH 5.5 wasfirst filtered through a GF/B filter and subsequently through a 0.45 μmfilter. The filtered sample was desalted using a Superdex G25 SP column(430 ml, 22×5 cm) equilibrated in 20 mM triethanolamine, pH 7.3. Theflow rate was 5 ml/min. The total volume after desalting was 150 ml.

The desalted sample was applied to a Source Q30 anion exchanger column(100 ml, 5×5 cm) equilibrated in 20 mM triethanolamine, pH 7.3. Thecolumn was washed with equilibration buffer until a stable baseline wasobtained. Lipase activity was eluted with a 420 ml linear gradient from0 to 0.35 M sodium chloride in equilibration buffer, flow rate 5 ml/min.Fractions of 10 ml were collected. Sodium acetate (100 μl of a 2Msolution) was added to each fraction to adjust pH to 5.5. Fractions26-32 (70 ml) were pooled.

To the pool from the anion exchange step was added ammonium sulfate to 1M and the sample was applied to a Source Phenyl HIC column (20 ml, 10×2cm) equilibrated in 20 mM sodium acetate (pH 5.5), 1 M ammonium sulfate.The column was washed with the equilibration buffer. Lipase was elutedwith a 320 ml linear gradient from 1 M to 0 M ammonium sulfate in 20 mMsodium acetate (pH 5.5), flow 1.5 ml/min. Fractions of 7.5 ml werecollected.

Fractions 33-41 were analyzed by SDS-PAGE using a NOVEX system withprecast gels. Both electrophoresis and silver staining of the gels weredone according to the manufacturer (Novex, San Diego, USA). (The samesystem was used for native electrophoresis and isoelectric focusing). Itwas found that fraction 40 and 41 contained lipase as the only protein.

2.3. Characterization of the purified lipase

(i) Determination of molecular weight

The apparent molecular weight of the native lipase was 37.7 kDa asmeasured by the above SDS-PAGE procedure. The purified lipase eluted ata molecular weight of 32.2 kDa from a Superose 12 gel filtration column(50 mM sodium phosphate, 0.2 M sodium chloride, pH 6.85, flow 0.65ml/min) and is therefore a monomer.

The molecular weight of the lipase was also determined bymatrix-assisted laser desorption ionisation (MALDI) by means of atime-of-flight (TOF) mass spectrometer (Voyager Bio-SpectrometryWorkstation, Perspective Biosystems). Samples were prepared by mixing0.7 μl of desalted lipase solution and 0.7 μl of a matrix solutioncontaining sinapic acid (3.5-dimethoxy-4-hydroxy cinnamic acid) in 70%acetonitrile (0.1% TFA, 10 mg/ml). 0.7 μl of the sample mixture wasplaced on top of a stainless steel probe tip and allowed to air-dryprior to introduction into the mass spectrometer. Spectra were obtainedfrom at least 100 laser shots and averaged to obtain a good signal tonoise ratio. The molecular mass for the lipase was found to be 30,384 Daand 30,310 Da by two independent analyses.

Digestion of the lipase with endo-β-N-acetyl-glucosamidase H (10 μl)from Streptomyces (Sigma) was carried out by adding 200 μl lipase andincubating at 37° C. for 2 hours. The digestion mixture was desaltedusing a VSWP filter and analyzed directly by MALDI mass spectrometry. Amajor component of deglycosylated lipase gave a mass of 29,339 Da and29,333 Da by two independent analyses. A minor component with a mass of29,508 Da was also observed. These values corresponds well to the latercalculated theoretical value of 28,939 Da based on the complete aminoacid sequence of the mature lipase.

(ii) Determination of the isoelectric point

The isoelectric point (pI) for the lipase was determined by isoelectricfocusing and was found to be 4.1.

A calculation of the pI based on the amino acid sequence as determinedin the following and shown as SEQ ID NO: 9 gave an estimated pI of 4.07.

(iii) Determination of temperature stability

Eppendorf tubes with 25 μl of purified lipase 3 plus 50 μl 100 mM sodiumacetate buffer (pH 5.0) were incubated for 1 hour in a water bath atrespectively 30, 40, 50, and 60° C. A control was treated in the sameway, but left at room temperature. After 1 hour the lipase 3 activitywas determined by the p-nitrophenyl acetate assay as described above.

The purified lipase had a good thermostability. It was found that thelipase maintained 60% of its activity after 1 hour at 60° C. 80% and 85%activity was maintained after 1 hour at 50° C. and 40° C. respectively.

(iv) Determination of pH stability

Purified lipase 3 (200 μl) was added to 5 ml of 50 mM buffer solutions:(sodium phosphate, pH 8.0, 7.0 and 6.0 and sodium acetate pH 5.0, 4.0and 3.5). The control was diluted in 5 ml of 4 mM sodium acetate pH 5.5.After four days at room temperature the residual activity was measuredby the Modified Food Chemical Codex assay for lipase activity asdescribed above. The lipase was very stable in the pH range from 4.0 to7.0 where it maintained about 100% activity relative to the control(Table 2.1). At pH 3.5 the lipase maintained 92% activity, and at pH 8.095% residual activity was maitained as compared to the control.

TABLE 2.1 pH stability of lipase 3 pH Activity (LUT/ml) Activity (%)Control (pH 5.5) 89.2 100 3.5 82.5 92 4.0 91.7 103 5.0 86.5 97 6.0 92.4104 7.0 90.6 102 8.0 84.4 95

EXAMPLE 3 Amino acid sequencing of lipase 3

Purified lipase enzyme was freeze-dried and 100 μg of the freeze-driedmaterial was dissolved in 50 μl of a mixture of 8 M urea and 0.4 Mammonium hydrogencarbonate, pH 8.4. The dissolved protein was denaturedand reduced for 15 minutes at 50° C. following overlay with nitrogen andaddition of 5 μl 45 mM dithiothreitol. After cooling to roomtemperature, 5 μl of 100 mM iodoacetamide was added for the cysteineresidues to be derivatized for 15 minutes at room temperature in thedark under nitrogen.

135 μl of water and 5 μg of endoproteinase Lys-C in 5 μl of water wasadded to the above reaction mixture and the digestion was carried out at37° C. under nitrogen for 24 hours. The resulting peptides wereseparated by reverse phase HPLC on a VYDAC C18 column (0.46×15 cm; 10μm; The Separation Group, California, USA) using solvent A: 0.1% TFA inwater and solvent B: 0.1% TFA in acetonitrile. Selected peptides wererechromatographed on a Develosil C18 column (0.46×10 cm, Novo Nordisk,Bagsværd, Denmark) using the same solvent system, prior to N-terminalsequencing. Sequencing was done using an Applied Biosystems 476Asequencer using pulsed-liquid fast cycles according to themanufacturer's instructions (Applied Biosystems, California, USA).

For direct N-terminal sequencing, the purified protein was passedthrough a Brownlee C2 Aquapore column (0.46×3 cm, 7 μm, AppliedBiosystems, California, USA) using the same solvent system as above.N-terminal sequencing was then performed as described above. As theprotein was not derivatized prior to sequencing, cysteine residues couldnot be determined.

The following peptide sequences were found:

N-terminal:Ser-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln-Trp-Ser-Ala-Ala-Ala-Tyr-X-Ser-Asn-Asn(SEQ ID NO:1)

Internal peptide 1: Val-His-Thr-Gly-Phe-Trp-Lys (SEQ ID NO:2)

Internal peptide 2:Ala-Trp-Glu-Ser-Ala-Ala-Asp-Glu-Leu-Thr-Ser-Lys-Ile-Lys (SEQ ID NO:3)

No further peptides could be purified from the HPLC fractionationassumingly because they were very hydrophobic and therefore tightlybound to the reverse phase column.

A search in SWISS-PROT database release 31 for amino acid sequences withhomology to the above peptides was performed and only three sequenceswere found.

All of the above peptides showed a low homology to the above knownsequences. Especially internal peptide 2 has very low homology to thethree lipases, (SEQ ID NO:10), (SEQ ID NO:11) and MDLA-(SEQ ID NO:12)from Rhizopus delamar (Haas and Berka, Gene, 1991, 109:107-113),Rhizomucor miehei (Boel et al., Lipids, 1988, 23:701-706) andPenicillium camenbertii (Yamaguchi et al., Gene, 1991, 103:61-67; Isobeand Nokihara, Febs. Lett., 1993, 320:101-106) respectively. Although thehomology was not very high it was possible to position the lipase 3peptides on these sequences as it is shown in the below Table 3.1.

TABLE 3.1 Alignment of lipase 3 peptides with known lipase sequences(SEQ ID NO:10) LIP_RHIDLMVSFISISQGVSLCLLVSSMMLGSSAVPVSGKSGSSNTAVSASDNAALPP 50 (SEQ ID NO:11)LIP_RHIMI MVLKQRANYLGFLIVFFTAFLV--EAVPIKRQSNSTVDS--------LPP 40 (SEQ IDNO:12) MDLA_PENCA MRLS-----------FFTAL------------------SAVASLGYALPG 21*              . ...                  .        ** (SEQ ID NO:1)N-Terminal            SVSTSTLDELQLFAQWSAAAYXSNN LIP_RHIDLLISSRCAPPSNKGSKSDLQAEPYNMQKNTEWYESHGGNLTSIGKRDDNLV 100 LIP_RHIMILIPSRTSAPSSSPSTTDPEAPAM----------SRNGPLPS----DVETK 76 MDLA_PENCAKLQSR------DVSTSELDQFEFWVQYAAASY------------------ 47. **      . *... .. LIP_RHIDLGGMTLDLPSDAPPISLSSSTNSASDGGKVVAATTAQIQEFTKYAGIAATA 150 LIP_RHIMIYGMALNATSYPDSV-----VQAMSIDGGIRAATSQEINELTYYTTLSANS 121 MDLA_PENCA-------------------------------------YEADYTAQVGDKL 60                                      * .  . .... LIP_RHIDLYCRSVVPGNKWDCVQCQKWVPDGKIITTFT-SLLSDTNGYVLRSDKQKTI 199 LIP_RHIMIYCRTVIPGATWDCIHCDA-TEDLKIIKTWS-TLIYDTNAMVARGDSEKTI 169 MDLA_PENCASCSKG------NCPEVEA--TGATVSYDFSDSTITDTAGYLAVDHTNSAV 102 *..       .* . .    . ..  ... . . **.. .  ....... (SEQ ID NO:2) Peptide1                                  VHTGFWK (SEQ ID NO:3) Peptide 2                                      AWESAADELTSK LIP_RHIDLYLVFRGTNSFRSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPV 249 LIP_RHIMIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVAT 219 MDLA_PENCAVLAFRGSYSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKE 152 ..***. * *. ..* .*   .    .*  .. ** ...  ... Peptide 2 IK (cont.)LIP_RHIDL VQEQLTAHPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVG 299LIP_RHIMI VLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQG 269MDLA_PENCA LKEVVAQNPNYELVVVGHSLGAAVATLAATDL--RGKGYPSAKLYAYA-- 198. .   . *.*.. *.*****.* * * . **  *.   .. .*  .. LIP_RHIDLGPRVGNPTFAYYVESTGIPFQRTVHKRDIVPHVPPQSFGFLHPGVESWIK 349 LIP_RHIMIQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWIT 319 MDLA_PENCASPRVGNAALAKYITAQGNNF-RFTHTNDPVPKLPLLSMGYVHVSPEYWIT 247 ****....* *. . *  . * ....* **..*  ..*..* . * **. LIP_RHIDLSGTSN-V-----QICTSEIETKDCSNSIVPFTSILD-HLSYF-DINEGSC 391 LIP_RHIMIDNSPETV-----QVCTSDLETSDCSNSIVPFTSVLD-HLSYF-GINTGLC 362 MDLA_PENCASPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDAGKG 297. . . *     .. ..... .. ... .*. . .. *. **  ...* LIP_RHIDL -------L 392LIP_RHIMI -------T 363 MDLA_PENCA PGLPFKRV 305

EXAMPLE 4 Isolation and purification of Aspergillus tubigensis genomicDNA

The Aspergillus tubigensis mutant strain was grown in PDB (Difco) for 72hours and the mycelium was harvested. 0.5-1 g of mycelium was frozen inliquid nitrogen and ground in a mortar. Following evaporation of thenitrogen, the ground mycelium was mixed with 15 ml of an extractionbuffer (100 nM Tris.HCl, pH 8.0, 50 mM EDTA, 500 mM NaCl, 10 mMβ-mercaptoe-thanol) and 1 ml 20% sodium dodecylsulfate. The mixture wasvigorously mixed and incubated at 65° C. for 10 min. 5 ml 3M potassiumacetate, (pH 5.1 adjusted with glacial acetic acid) was added and themixture further incubated on ice for 20 min. The cellular debris wasremoved by centrifugation for 20 min. at 20,000×g and 10 ml isopropanolwas added to the supernatant to precipitate (30 min at −20° C.) theextracted DNA. After further centrifugation for 15 min at 20,000×g, theDNA pellet was dissolved in 1 ml TE (10 mM Tris.HCl pH 8.0, 1 mM EDTA)and precipitated again by addition of 0.1 ml 3 M NaAc, pH 4.8 and 2.5 mlethanol. After centrifugation for 15 min at 20,000×g the DNA pellet waswashed with 1 ml 70% ethanol and dried under vacuum. Finally, the DNAwas dissolved in 200 μl TE and stored at −20° C.

EXAMPLE 5 The generation of a fragment of the putative gene coding forlipase 3 using PCR

To obtain a fragment of the putative gene (in the following referred toas the lipA gene) as a tag to isolate the complete gene, a PCRamplification procedure based on the information in the isolated peptidesequences was carried out.

Degenerated primers for PCR amplification of a fragment of the lipasegene were designed based on the amino acid sequences of the isolatedpeptides. The following three PCR primers were synthesised:

C035: TTC CAR AAN CCN GTR TGN AC (SEQ ID NO:4) 20 mer 256 mixture, basedon peptide 1 sequence VHTGFWK (SEQ ID NO:2) (Reversed).

C036: CAR YTN TTY GCN CAR TGG (SEQ ID NO:5) 18 mer 256 mixture, based onthe N-terminal sequence QLFAQW.

C037: GCV GCH SWY TCC CAV GC (SEQ ID NO:6)

17 mer 216 mixture, based on internal peptide 2 sequence AWESAA(reversed).

The oligonucleotides were synthesised on a Applied Biosystems model 392DNA/RNA Synthesizer. To reduce the degree of degeneracy the rare Alacodon GCA and the Ser codon TCA have been excluded in design of primerC037.

With these primers the desired fragments were amplified by PCR. Usingthese primers it was expected that a fragment of about 300 bp should beamplified provided there are no introns in the fragment.

The following PCR reactions were set up in 0.5 ml PCR tubes to amplify aputative lipA fragment:

1. 0.5 μg total genomic DNA,

100 pmol primer C036,

100 pmol primer C037,

10 μl PCR Buffer II (Perkin Elmer),

6 μl 25 mM MgCl₂,

2 μl dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP),

2 units Amplitaq polymerase (Perkin Elmer), and water to a total volumeof 100 μl.

2. 0.5 μg total genomic DNA,

100 pmol primer C035,

100 pmol primer C036,

10 μl PCR Buffer II (Perkin Elmer),

6 μl 25 mM MgCl₂,

2 μl dNTP mix (10 mM DATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP),

2 units Amplitaq polymerase (Perkin Elmer), and water to a total volumeof 100 μl.

The reactions were performed using the following program:

94° C. 2 min

94° C. 1 min)

40° C. 1 min)

72° C. 1 min) These three steps were repeated for 30

72° C. 5 min cycles

5° C. SOAK

The PCR amplifications were performed in a MJ Research Inc. PTC-100Thermocycler.

In reaction 1, three distinct bands of about 300, 360 and 400 bp,respectively could be detected. These bands were isolated and clonedusing the pT7-Blue-T-vector kit (Novagene). The sizes of these fragmentis in agreement with the expected size provided that the fragmentcontains 0, 1 or 2 introns, respectively.

The three fragments were sequenced using a “Thermo Sekvenase fluorescentlabelled primer cycle sequencing Kit” (Amersham) and analyzed on a ALFsequencer (Pharmacia) according to the instructions of the manufacturer.The fragment of about 360 bp contained a sequence that was identified asa lipase and, as it contained the part of the N-terminal distal to thesequence used for primer design, it was concluded that the desired lipAgene fragment was obtained.

The sequence of the about 360 bp PCR fragment (SEQ ID NO:7) is shown inthe following Table 5.1 (SEQ ID NO: 13). The four amino acid fragmentsof Table 5.1 are contained in SEQ ID NOS; 14, 15, 16, and 17. Thepeptide sequence used for primer design is underlined. The remainingpart of the N-terminal sequence is doubly underlined.

TABLE 5.1 PCR-generated putative lipA sequence        10        20        30        40        50        60         |         |         |         |         |         |tacccggggntccgattCAGTTGTTCGCGCAATGGTCTGCCGCAGCTTATTGCTCGAATA                  O  L  F  A  O  W  S  A  A  A  Y  C  X  N        70        80        90       100       110       120         |         |         |        |         |         |ATATCGACTCGAAAGAVTCCAACTTGACATGCACGGCCAACGCCTGTCCATCAGTCGAGG N  I  D  S  K  X  S  N  L  T  C  T  A  N  A  C  P  S  V  E       130       140       150       160       170       180         |         |         |         |         |         |AGGCCAGTACCACGATGCTGCTGGAGTTCGACCTGTATGTCACTCAGATCGCAGACATAGE  A  S  T  T  M  L  L  E  F  D  L  Y  V  T  Q  I  A  D  I       190       200       210       220       230       240         |         |         |         |         |         |AGCACAGCTAATTGAACAGGACGAACGACTTTTGGAGGCACAGCCGGTTTCCTGGCCGCGE  H  S  -  L  N  R  T  N  D  F  W  R  H  S  R  F  P  G  R       250       260       270       280       290       300         |         |         |         |         |         |GACAACACCAACAAGCGGCTCGTGGTCGCCTTCCGGGGAAGCAGCACGATTGAGAACTGGG  Q  H  Q  Q  A  A  R  G  R  L  P  G  K  Q  H  D  -  E  L       310       320       330          |         |         |ATTGCTAATCYTGACTTCATCCTGGRAGATAACG D  C  -  X  -  L  H  P  X  R  -

The finding of this sequence permitted full identification of the PCRfragment as part of the lipA gene. The stop codon found in the readingframe can be caused either by a PCR or a reading error or there can bean intron encoded in the fragment as a consensus intron start and endingsignal (shown in bold). If the putative intron is removed a shift inreading frame will occur. However, an alignment of the deduced aminoacid sequence and the fungal lipases shown in Table 3.1 suggested thatthe fragment was part of the desired gene.

EXAMPLE 6 Cloning and characterisation of the lipA gene

(i) Construction of an Aspergillus tubigensis genomic library

Aspergillus tubigensis genomic DNA was digested partially with Tsp5091(New England Biolabs Inc.). 10 μg DNA was digested in 100 μl reactionmixture containing 2 units Tsp5091. After 5, 10, 15 and 20 minutes 25 μlwas removed from the reaction mixture and the digestion was stopped byaddition of 1 μl 0.5 M EDTA, pH 8.0. After all four reactions had beenstopped, the samples were run on a it agarose gel in TAE buffer (10×TAEstock containing per liter: 48.4 g Trizma base, 11.5 ml glacial aceticacid, 20 ml 0.5 M EDTA pH 8.0). HindIII-digested phage Lambda DNA wasused as molecular weight marker (DNA molecular weight marker II,Boehringer, Mannheim). Fragments of a size between about 5 and 10 kbwere cut out of the gel and the DNA fragments were purified using GeneClean II Kit (Bio-101 Inc.). The purified fragments were pooled and 100ng of the pooled fragments were ligated into 1 μg EcoRI-digested anddephosphorylated ZAP II vector (Stratagene) in a total volume of 5 μl. 2μl of this volume was packed with Gigapack II packing extract(Stratagene) which gave a primary library of 650,000 pfu.

E. coli strain XL1-Blue-MRF (Stratagene) was infected with 5×50,000 pfuof the primary library. The infected bacteria were mixed with topagarose (as NZY plates but with 6 g agarose per liter instead of theagar) and plated on 5 NZY plates (13 cm). After incubation at 37° C. for7 hours, 10 ml SM buffer (per liter: 5.8 g NaCl, 2.0 g MgCl₂.7H₂O, 50 ml1 M Tris.HCl pH 7.5, 5.0 ml of 2% (w/v) gelatine) and incubatedovernight at room temperature with gently shaking. The buffer containingwashed-out phages was collected and pooled. 5% chloroform was added andafter vigorous mixing the mixture was incubated 1 hour at roomtemperature. After centrifugation for 2 minutes at 10,000×g the upperphase containing the amplified library was collected anddimethylsulphoxide was added to 7%. Aliquots of the library was takenout in small tubes and frozen at −80° C. The frozen library contained2.7×10⁹ pfu/ml with about 6% without inserts.

(ii) Screening of the Aspergillus tubigensis library

2×50.000 pfu were plated on large (22×22 cm) NZY plates containing amedium containing per liter: 5 g NaCl, 2 g MgSO₄.7H₂O, 5 g yeastextract, 10 g casein hydrolysate, 15 g agar, pH adjusted to 7.5 withNaOH. The medium was autoclaved and cooled to about 60° C. and pouredinto the plates. Per plate was used 240 ml of medium.

The inoculated NZY plates were incubated overnight at 37° C. and plaquelifts of the plates were made. Two lifts were made for each plate onHybond N (Amersham) filters. The DNA was fixed using UV radiation for 3min. and the filters were hybridized as described in the followingusing, as the probe, the above PCR fragment of about 360 bp that waslabelled with ³²P-dCTP using Ready-to-Go labelling kit (Pharmacia).

The filters were prehybridised for one hour at 65° C. in 25 mlprehybridisation buffer containing 6.25 ml 20×SSC (0.3 M Na₃citrate, 3 MNaCl), 1,25 ml 100×Denhard solution, 1.25 ml 10% SDS and 16.25 ml water.150 μl 10 mg/ml denatured Salmon sperm DNA was added to theprehybridization buffer immediately before use. Followingprehybridization, the prehybridisation buffer was discarded and thefilters hybridised overnight at 65° C. in 25 ml prehybridisation bufferwith the radiolabelled PCR fragment.

Next day the filters were washed according to the following procedure:2×15 min. with 2×SSC+0.1% SDS, 15 min. with 1×SSC+0.1% SDS and 10 min.with 0.1×SSC+0.1% SDS.

All washes were done at 65° C. The sheets were autoradiographed for 16hours and positive clones were isolated. A clone was reckoned aspositive only if there was a hybridisation signal on both plaque liftsof the plate in question.

Seven putative clones were isolated and four were purified by plating onsmall petri dishes and performing plaque lifts essentially as describedabove.

The purified clones were converted to plasmids using an ExAssist Kit(Stratagene).

Two sequencing primers were designed based on the about 360 bp PCRfragment. The sequencing primers were used to sequence the clones and apositive clone with the lipA gene encoding lipase 3 was found. Theisolated positive clone was designated pLIP4.

(iii) Characterisation of the pLIP4 clone

A restriction map of the clone was made. The above 360 bp PCR fragmentcontained a SacII site and as this site could be found in the genomicclone as well this site facilitated the construction of the map. Therestriction map showing the structure of pLIP4 is shown in FIG. 1. Therestriction map shows that the complete gene is present in the clone.Additionally, since promoter and terminator sequences are present, itwas assumed that all the important regions is present in the clone.

A sample of Escherichia coli strain DH5α containing pLIP4 was depositedin accordance with the Budapest Treaty with The National Collections ofIndustrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive,Aberdeen, Scotland, United Kingdom, AB2 1RY on Feb. 24, 1997 under theaccession number NCIMB 40863.

The gene was sequenced using cycle sequencing and conventionalsequencing technology. The complete sequence is shown below in Table 6.1(SEQ ID NO: 18). The sequence has been determined for both strands forthe complete coding region and about 100 bp upstream and downstream ofthe coding region. The sequences downstream to the coding region haveonly been determined on one strand and contains a few uncertainties. Inthe sequence as shown below, the intron ences are indicated as lowercaseletters and the N-terminal and the two internal peptides (peptide 1 andpeptide 2) are underlined:

TABLE 6.1. The DNA seauence for the lipA gene and f1anking sequences(SEQ ID NO:18) 1CCNDTTAATCCCCCACCGGGGTTCCCGCTCCCGGATGGAGATGGGGCCAAAACTGGCAAC 61CCCCAGTTGCGCAACGGAACAACCGCCGACCCGGAACAAAGGATGCGGATGAGGAGATAC 121GGTGCCTGATTGCATGGCTGGCTTCATCTGCTATCGTGACAGTGCTCTTTGGGTGAATAT 181TGTTGTCTGACTTACCCCGCTTCTTGCTTTTTCCCCCCTGAGGCCCTGATGGGGAATCGC 241GGTGGGTAATATGATATGGGTATAAAAGGGAGATCGGAGGTGCAGTTGGATTGAGGCAGT 301GTGTGTGTGTGCATTGCAGAAGCCCGTTGGTCGCAAGGTTTTGGTCGCCTCGATTGTTTG 361TATACCGCAAGATGTTCTCTGGACGGTTTGGAGTGCTTTTGACAGCGCTTGCTGCGCTGG            M  F  S  G  R  F  G  V  L  L  T  A  L  A  A  L 421GTGCTGCCGCGCCGGCACCGCTTGCTGTGCGGAgtaggtgtgcccgatgtgagatggttgG  A  A  A  P  A  P  L  A  V  R 481gatagcactgatgaagggtgaatagGTGTCTCGACTTCCACGTTGGATGAGTTGCAATTG                         S  V  S  T  S  T  L  D  E  L  O  L 541TTCGCGCAATGGTCTGCCGCAGCTTATTGCTCGAATAATATCGACTCGAAAGACTCCAAC F  A  O  W  S  A  A  A  Y  C  S  N  N  I  D  S  K  D  S  N 601TTGACATGCACGGCCAACGCCTGTCCATCAGTCGAGGAGGCCAGTACCACGATGCTGCTG L  T  C  T  A  N  A  C  P  S  V  E  E  A  S  T  T  M  L  L 661GAGTTCGACCTgtatgtcactcagatcgcagacatagagcacagctaatttgaacagGAC E  F  D  L                                               T  721GAACGACTTTGGAGGCACAGCCGGTTTCCTGGCCGCGGACAACACCAACAAGCGGCTCGT  N  D  F  G  G  T  A  G  F  L  A  A  D  N  T  N  K  R  L  V 781GGTCGCCTTCCGGGGAAGCAGCACGATTGAGAACTGGATTGCTAATCTTGACTTCATCCT  V  A  F  R  G  S  S  T  I  E  N  W  I  A  N  L  D  F  I  L 841GGAAGATAACGACGACCTCTGCACCGGCTGCAAGGTCCATACTGGTTTCTGGAAGGCATG  E  D  N  D  D  L  C  T  G  C  K  V  H  T  G  F  W  K  A  W 901GGAGTCCGCTGCCGACGAACTGACGAGCAAGATCAAGTCTGCGATGAGCACGTATTCGGG  E  S  A  A  D  E  L  T  S  K  I  K  S  A  M  S  T  Y  S  G 961CTATACCCTATACTTCACCGGGCACAGTTTGGGCGGCGCATTGGCTACGCTGGGAGCGAC  Y  T  L  Y  F  T  G  H  S  L  G  G  A  L  A  T  L  G  A  T 1021AGTTCTGCGAAATGACGGATATAGCGTTGAGCTGgtgagtccttcacaaaggtgatggag  V  L  R  N  D  G  Y  S  V  E  L 1081cgacaatcgggaacagacagtcaatagTACACCTATGGATGTCCTCGAATCGGAAACTAT                            Y  T  Y  G  C  P  R  I  G  N  Y 1141GCGCTGGCTGAGCATATCACCAGTCAGGGATCTGGGGCCAACTTCCGTGTTACACACTTG A  L  A  E  H  I  T  S  Q  G  S  G  A  N  F  R  V  T  H  L 1201AACGACATCGTCCCCCGGGTGCCACCCATGGACTTTGGATTCAGTCAGCCAAGTCCGGAA N  D  I  V  P  R  V  P  P  M  D  F  G  F  S  Q  P  S  P  E 1261TACTGGATCACCAGTGGCAATGGAGCCAGTGTCACGGCGTCGGATATCGAAGTCATCGAG Y  W  I  T  S  G  N  G  A  S  V  T  A  S  D  I  E  V  I  E 1321GGAATCAATTCAACGGCGGGAAATGCAGGCGAAGCAACGGTGAGCGTTGTGGCTCACTTG G  I  N  S  T  A  G  N  A  G  E  A  T  V  S  V  V  A  H  L 1381TGGTACTTTTTTGCGATTTCCGAGTGCCTGCTATAACTAGACCGACTGTCAGATTAGTGG W  Y  F  F  A  I  S  E  C  L  L  - 1441ACGGGAGAAGTGTACATAAGTAATTAGTATATAATCAGAGCAACCCAGTGGTGGTGATGG 1501TGGTGAAAGAAGAAACACATTGAGTTCCCATTACGKAGCAGWTAAAGCACKTKKGGAGGC 1561GCTGGTTCCTCCACTTGGCAGTTGGCGGCCATCAATCATCTTTCCTCTCCTTACTTTCGT 1621CCACCACAACTCCCATCCTGCCAGCTGTCGCATCCCCGGGTTGCAACAACTATCGCCTCC 1681GGGGCCTCCGTGGTTCTCCTATATTATTCCATCCGACGGCCGACGTTTCACCCTCAACCT 1741GCGCCGCCGCAAAATCTCCCCGAGTCGGTCAACTCCCTCGAACCGCCGCCCGCATCGACC 1801TCACGACCCCGACCGTCTGYGATYGTCCAACCG

(iv) Analysis of the sequence of the complete gene

The peptide sequences obtained could all be found in the deduced aminoacid sequence (see Table 5.1) which confirms again that the sequencefound is the sequence of the lipase 3 gene. The gene was designatedlipA.

The amino acid sequence was aligned with the three fungal lipases usedto align the peptide sequences. The alignment is shown in Table 6.2.

TABLE 6.2 A1ignment of the lipase 3 sequence with known fungal lipasesLIPASE3 MFSG-------------RFGVLL-----------------------TALAA 15MDLA_PENCA MRLS-------------FFTAL------------------------SAVAS 14LIP_RHIDL MVSFISISQGVSLCLLVSSMMLGSSAVPVSGKSGSSNTAVSASDNAALPP 50LIP_RHIMI MVLKQRANYLGFLIVFFTAFLV--EAVPIKRQSNSTVDS--------LPP 40*                . .                           ... LIPASE3L-------------------------------------------------- 16 MDLA_PENCAL-------------------------------------------------- 15 LIP_RHIDLLISSRCAPPSNKGSKSDLQAEPYNMQKNTEWYESHGGNLTSIGKRDDNLV 100 LIP_RHIMILIPSRTSAPSSSPSTTDPEAPAM----------SRNGPLPS----DVETK 76 * LIPASE3--------GAAAPAPLA-----------VRSVSTSTLDELQLFAQWSAAA 47 MDLA_PENCA--------GYALPGKLQ-----------SRDVSTSELDQFEFWVQYAAAS 46 LIP_RHIDLGGMTLDLPSDAPPISLSSSTNSASDGGKVVAATTAQIQEFTKYAGIAATA 150 LIP_RHIMIYGMALNATSYPDSV-----VQAMSIDGGIRAATSQEINELTYYTTLSANS 121        . . .                 ....  ....  ..  .*.. LIPASE3YCSNNIDSK-DSNLTCTANACPSVEEASTTMLLEFDLTNDFGGTAGFLAA 96 MDLA_PENCAYYEADYTAQVGDKLSCSKGNCPEVEATGATVSYDFS-DSTITDTAGYIAV 95 LIP_RHIDLYCRSVVP---GNKWDCVQ--CQKWVPDGKIIT---TFTSLLSDTNGYVLR 192 LIP_RHIMIYCRTVIP---GATWDCIH--CDA-TEDLKIIK---TWSTLIYDTNAMVAR 162*. .      ... .*    *     .  ..    . .. . .*.. . LIPASE3DNTNKRLVVAFRGSSTIENWIANLDFILEDNDDLCTGCKVHTGFWKAWES 146 MDLA_PENCADHTNSAVVLAFRGSYSVRNWVADATFV-HTNPGLCDGCLAELGFWSSWKL 144 LIP_RHIDLSDKQKTIYLVFRGTNSFRSAITDIVFNFSDYKPV-KGAKVHAGFLSSYEQ 241 LIP_RHIMIGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPV-SGTKVHKGFLDSYGE 211..... . ..***. .  . ...  *   .   . .*  .. ** ... LIPASE3AADELTSKIKSAMSTYSGYTLYFTGHSLGGALATLGATVL--RNDGY-SV 193 MDLA_PENCAVRDDIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDL--RGKGYPSA 192 LIP_RHIDLVVNDYFPVVQEQLTAHPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPK 291 LIP_RHIMIVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSS 261. ..    . .     ..*..  .*****.* * * .  *  *.    . LIPASE3ELYTY--GCPRIGNYALAEHITSQGSGANFRVTHLNDIVPRVPPMDFGFS 241 MDLA_PENCAKLYAY--ASPRVGNAALAKYITAQGN--NFRFTHTNDPVPKLPLLSMGYV 238 LIP_RHIDLNLSIFTVGGPRVGNPTFAYYVESTGIPFQ-RTVHKRDIVPHVPPQSFGFL 340 LIP_RHIMINLFLYTQGQPRVGDPAFANYVVSTGIPYR-RTVNERDIVPHLPPAAFGFL 310.*  .  . **.*. ..* .. . *   . * .. .* **..*  ..*. LIPASE3QPSPEYWITSGNGASVTASDIEVIEGINSTAGNAGEATVSVV---AHLWY 288 MDLA_PENCAHVSPEYWITSPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWY 288 LIP_RHIDLHPGVESWIKSGTSN-VQICTSEIE------TKDCSNSIVPFTSILDHLSY 383 LIP_PHINIHAGEEYWITDNSPETVQVCTSDLE------TSDCSNSIVPFTSVLDHLSY 354. . * **.. . . *  .. ..       . . ...    .   .*. . LIPASE3FFAISECL--------L 297 MDLA_PENCA FVQVDAGKGPGLPFKPV 305 LIP_RHIDLF-DINEGSC-------L 392 LIP_RHIMI F-GINTGLC-------T 363 *  ...

The above alignment shows that lipase 3 is homologous to the knownlipase sequences but that the homology is not very high. Deletions orinsertions in the lipase 3 sequence was not observed when comparing thesequence with these three lipases. This strengthens the probability thatthe putative introns have been identified correctly.

A search in SWISS-PROT release 31 database was performed and it did notlead to further sequences with higher homology than that to the aboveknown lipases (Table 6.3).

The sequence with highest homology is a mono- diacyl lipase fromPenicillium camembertii where the identity is found to 42%. However theC- terminal of lipase 3 resembles the 2 lipases from Zygomycetes(Rhizopus and Rhizomucor) and not the P. camembertii enzyme.

TABLE 6.3 A1ignment of coding seauences of the lipA gene and gene codingfor mono-diacyl lipase from Penicillium camemberti LIPASE3-MFSGRFGVLLTALAALGAAAPAPLAVRSVSTSTLDELQLFAQWSAAAYCS -50|    |  |  | | || | |  |  | |||| ||      |  || | MDLA_PENCA-MRLSFFTAL-SAVASLGYALPGKLQSRDVSTSELDQFEFWVQYAAASYYE -49 LIPASE3-NNIDSK-DSNLTCTANACPSVEEASTTMLLEFDLTNDFGGTAGFLAADNT -99          | |    || ||    |    |        |||  | | | MDLA_PENCA-ADYTAQVGDKLSCSKGNCPEVEATGATVSYDFS-DSTITDTAGYIAVDHT -98 LIPASE3-NKRLVVAFRGSSTIENWIANLDFILEDNDDLCTGCKVHTGFWKAWESAAD -149|   | |||||    || |   |    |  || ||    |||  |    | MDLA_PENCA-NSAVVLAFRGSYSVRNWVADATFV-HTNPGLCDGCLAELGFWSSWKLVRD -147 LIPASE3-ELTSKIKSAMSTYSGYTLYFTGHSLGGALATLGATVLRNDGY-SVELYTY -198      |        | |   ||||| | ||| || ||  || |  || | MDLA_PENCA-DIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAY -197 LIPASE3-GCPRIGNYALAEHITSQGSGANFRVTHLNDIVPRVPPMDFGFSQPSPEYW -248  || || |||  || ||   ||| || || ||  |    |    ||||| MDLA_PENCA-ASPRVGNAALAKYITAQGN--NFRFTHTNDPVPKLPLLSMGYVHVSPEYW -245 LIPASE3-ITSGNGASVTASDIEVIEGINSTAGNAGEATVSVV---AHLWYFFAISEC -295||| | | |  ||| || |  |  || |          || ||| MDLA_PENCA-ITSPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDAG -295 LIPASE3-L--------L -297 MDLA_PENCA- KGPGLPFKRV -305 Identity: 126 amino acids(42.42%)

The N-terminal of the mature lipase has been determined by N-terminalsequencing to be the serine residue No. 28 of the lipase 3 precursor(SEQ ID NO:9) as shown in Table 6.4 below. Hence the amino acids No. 1to No. 27 is the signal sequence.

TABLE 6.4 Amino acid sequence of the precursor of lipase 3        5        10        15        20        25        30        |         |         |         |         |         | 1 M F S G RF G V L L T A L A A L G A A A P A P L A V R S V S 31 T S T L D E L Q L FA Q W S A A A Y C S N N I D S K D S N L 61 T C T A N A C P S V E E A S TT M L L E F D L T N D F G G T 91 A G F L A A D N T N K R L V V A F R G SS T I E N W I A N L 121 D F I L E D N D D L C T G C K V H T G F W K A WE S A A D E 151 L T S K I K S A M S T Y S G Y T L Y F T G H S L G G A LA T 181 L G A T V L R N D G Y S V E L Y T Y G C P R I G N Y A L A E 211H I T S Q G S G A N F R V T H L N D I V P R V P P M D F G F 241 S Q P SP E Y W I T S G N G A S V T A S D I E V I E G I N S 271 T A G N A G E AT V S V V A H L W Y F F A I S E C L L Number of residues: 297.

Residues 167-176 are recognised as a common motif for the serine lipases(PROSITE). The crystal structure for the Rhizomucor miehei serine lipasehas been examined and the residues in the active site identified (Bradyet al., Nature, 1990, 343:767-770; Derewanda et al., J. Mol. Biol.,1992, 227:818-839). The active site residues of R. miehei lipase haveall been conserved in all the lipases and correspond to the followingresidues in lipase 3: serine 173, aspartic acid 228 and histidine 285.

Lipase 3 contains 7 cysteine residues. Four of these are conserved inthe P. camembertii lipase where they form disulphide bonds (Isobe andNokuhara, Gene, 1991, 103:61-67). This corresponds to disulphide bondsbetween residue 62-67 and 131-134. In addition, two cysteine residuesare homologous to two C residues which forms an additional disulphidebond in Rhizopus and Rhizomucor lipases corresponding to residues49-295.

Two putative N-glycosylation sites were found in lipase 3 in position 59and 269. Neither of these are conserved in the other fungal lipases.

EXAMPLE 7 Transformation of Aspergillus tubigensis and overexpression oflipase 3 in A. tubigensis

The protocol for transformation was based on the teachings of Buxton etal. (Gene, 1985, 37:207-214), Daboussi et al (Curr. Genet., 1989,15:453-456) and Punt and van den Hondel, (Meth. Enzym., 1992,216:447-457).

A multicopy lipA strain was produced by transforming the pLIP4 plasmidinto Aspergillus tubigensis strain 6M 179 using cotransformation with ahygromycin resistant marker plasmid.

A screening procedure used to visualise fungal lipase after ultrathinlayer isoelectric focusing was adapted to screen Aspergillus tubigensistransformants grown on agar plates. Screening of lipase producers onagar plates was done using 2% olive oil as the substrate for the enzyme(lipase) as well as the inducer for the lipase promoter. In addition,the plates contained a fluorescent dye, Rhodamine B. In the presence ofolive oil, the transformants will be induced to secrete lipase. Thelipase secreted into the agar plate will hydrolyse the olive oil causingthe formation of orange fluorescent colonies that is visible upon UVradiation (350 nm). The appearence of fluorescent colonies was generallymonitored after 24 hours of growth. After several days of growth, thelipase producing strains could be identified as orange fluorescentstrains that are visible by eye. Under this plate screening condition,the untransformed strain gave no background fluorescence and appeared asopaque pink colonies.

Sixteen transformants that showed orange fluorescent halos werecultivated for 8 days in shake flasks containing 100 ml of minimalmedium supplemented with 1% olive oil, 0.5% yeast extract and 0.2%casamino acids. The amount of lipase secreted was quantified by applying10 μl of cell-free culture supernatant into holes punched in olive oil-Rhodamine B agar plates and incubating the plates overnight at 37° C.Five transformants with higher lipase production were found.

The cell-free culture supernatants from the five transformants weredesalted using NAP 5 columns (Pharmacia) and equilibrated in 1M ammoniumsulfate (50 mM sodium acetate, pH 5.5). The desalted culturesupernatants were fractionated by hydrophobic interaction chromatography(HIC) on a Biogel Phenyl-5 PW column (Biorad). Elution was done by adescending salt gradient of 1M to 0 M ammonium sulfate (20 mM sodiumacetate, pH 5.5). A single discrete protein peak was observed afterfractionation. The area of the protein peaks were calculated among thedifferent transformants and compared with the untransformed strain. Thebest transformant showed a 62-fold increase in the amount of lipaseafter HIC fractionation. A chromatogram of the HIC fractionated culturesupernatant of this transformant is shown in FIG. 3 and a similarchromatogram for the untransformed strain is shown in FIG. 4.

The fraction containing the transformed lipase was freeze-dried. Thetransformed lipase was carboxymethylated and subjected to N-terminalamino acid sequencing of the first 15 amino acids and it was found thatthe sequence of the recombinant lipase was exactly the same as thenative lipase indicating correct signal sequence cleavage.

The different lipase fractions collected after HIC were separated on a12% Tris-Glycine SDS gel and silver staining revealed one protein band,confirming the homogeneity of the fractions. In addition, the crudeextract showed a major lipase band as the only band that accumulated inthe culture supernatant in very high amounts when the fungus wascultured in the olive oil-containing medium.

The recombinant lipase was analysed by matrix-assisted laser desorptionionisation (MALDI) by means of a time-of-flight (TOF) mass spectrometeras described hereinbefore. The molecular weight of the recombinantlipase was 32,237 Da.

Detection of N-linked oligosaccharides was achieved by digestion of thelipase with endo-β-N-acetyl-glucosamidase H from Streptomyces (Sigma).Digestion of recombinant lipase secreted into the growth medium alteredthe mobility of the band seen on SDS-PAGE which moved as a single bandwith a molecular mass of about 30 kDa.

Deglycosylated recombinant lipase generated by digestion withendoglycosidase and analysed directly by MALDI mass spectrometry gave amolecular weight of the polypeptide backbone of 29,325 Da.

C. BAKING EXPERIMENTS EXAMPLE 8 Baking Experiments Using Lipase 3

8.1. Baking procedures and analytical methods

(i) Baking procedure for Danish toast bread

Flour (Danish reform flour) 2000 g, dry yeast 30 g, salt 30 g and watercorresponding to 400 Brabender units+3%, was kneaded in a Hobart Mixerwith hook for 2 min. at low speed and 10 min. at high speed. Doughtemperature after kneading was 25° C. Resting time was 10 min. at 30° C.The dough was scaled 750 g per dough and rested again for 5 min at 33°C. and 85% RH. After moulding on a Glimik moulder, the dough wereproofed in tins for 50 min at 33° C. and baked in a Wachtel oven for 40min at 220° C. with steam injection for 16 sec. After cooling, the breadwas scaled and the volume of the bread was measured by the rape seeddisplacement method. The specific volume is calculated by dividing thebread volume (ml) by the weight (g) of the bread.

The crumb was evaluated subjectively using a scale from 1 to 5 where1=coarsely inhomogeneous and 5=nicely homogeneous.

Three breads baked in tins with lid were stored at 20° C. and used forfirmness measurements and pore measurements by means of an ImageAnalyzer.

(ii) Baking procedure for Danish rolls

Flour (Danish reform) 1500 g, compressed yeast 90 g, sugar 24 g, salt 24g and water corresponding to 400 Brabender units−2% were kneaded in aHobart mixer with hook for 2 min. at low speed and 9 min at high speed.After kneading, the dough temperature was 26° C. The dough was scaled1350 g. After resting for 10 min. at 30° C., the dough was moulded on aFortuna moulder after which the dough was proofed for 45 min. at 34° C.and baked in a Bago oven for 18 min. at 220° C. with steam injection for12 sec. After cooling, the rolls were scaled and the volume of the rollswas measured by the rape seed displacement method. Specific volume iscalculated as described above.

(iii) Determination of pore homogeneity

The pore homogeneity of the bread was measured by means of an imageanalyzer composed of a standard CCD-video camera, a video digitiser anda personal computer with WinGrain software. For every bread, the resultsof pore diameter in mm and pore homogeneity were calculated as anaverage of measurements from 10 slices of bread. The pore homogeneitywas expressed in % of pores that are larger than 0.5 times the averageof pore diameter and smaller than 2 times the average diameter.

(iv) Determination of firmness

The firmness of bread, expressed as N/dm², was measured by means of anInstron UTM model 4301 connected to a personal computer. The conditionsfor measurement of bread firmness were:

Load Cell Max. 100 N Piston diameter 50 mm Cross head speed 200 mm/minCompression 25% Thickness of bread slice 11 mm

The result was an average of measurements on 10 bread slices for evertbread.

(v) Determination of gluten index

Gluten index was measured by means of a Glutomatic 2200 from PertenInstruments (Sweden). Immediately after proofing, 15 g of dough wasscaled and placed in the Glutomatic and washed with 500 ml 2% NaClsolution for 10 min. The washed dough was transferred to a Gluten IndexCentrifuge 2015 and the two gluten fractions were scaled and the glutenindex calculated according to the following equation:

Gluten index=(weight of gluten remaining on the sieve×100)/total weightof gluten

(vi) Extraction of lipids from dough

30 g of fully proofed dough was immediately frozen and freeze-dried. Thefreeze-dried dough was milled in a coffee mill and passed through a 235μm screen. 4 g freeze-dried dough was scaled in a 50 ml centrifuge tubewith screw lid and 20 ml water saturated n-butanol (WSB) was added. Thecentrifuge tube was placed in a water bath at a temperature of 100° C.for 10 min. after which the tubes were placed in a Rotamix and turned at45 rpm for 20 min. at ambient temperature. The tubes were again placedin the water bath for 10 min. and turned on the Rotamix for another 30min. at ambient temperature.

The tubes were centrifuged at 10,000×g for 5 min. 10 ml of thesupernatant was pipetted into a vial and evaporated to dryness undernitrogen cover. This sample was used for HPLC analysis.

A similar sample was fractionated on a Bond Elut Si (Varian 1211-3036).The non-polar fraction was eluted with 10 mlcyclohexan:isopropanol:acetic acid (55:45:1) and evaporated to dryness.This sample was used for GLC analysis.

(vii) HPLC analysis

Column: LiChrospher 100 DIOL 5 μm (Merck art. 16152) 250×4 mm with awater jacket of a temperature of 50° C.

Mobile phases:

A: heptan:isopropanol:n-butanol:tetrahydrofuran:isooctan:water(64.5:17.5:7:5:5:1)

B: isopropanol:n-butanol:tetrahydrofuran:isooctan:water (73:7:5:5:10)

The mobile phases contained 1 mmol trifluoroacetic acid per 1 mobilephase and were adjusted to pH 6.6 with ammonia.

Pump: Waters 510 equipped with a gradient controller.

Gradient: Flow (ml/min) Time (min) A (%) B (%) 1.0 0 100 0 1.0 25 0 1001.0 30 0 100 1.0 35 100 0 1.0 40 100 0

Detector: CUNOW DDL21 (evaporative light-scattering); temperature 100°C.; voltage: 600 volt; air flow: 6.0 l/min.

Injector: Hewlett Packard 1050; injection volume: 50 μl.

The samples for analysis were dissolved in 5 ml chloro-form:methanol(75:25), sonicated for 10 min and filtered through a 0.45 μm filter.

(viii) GLC analysis

Perkin Elmer 8420 Capillary Gas Chromatograph equipped with WCOT fusedsilica column 12.5 m×0.25 mm coated with 0.1 μm stationary phase of 5%phenyl-methyl-silicone (CP Sil 8 CB from Crompack).

Carrier: Helium

Injection: 1.5 μl with split

Detector: FID 385° C.

Oven program: 1 2 3 4 Oven temperature, ° C. 80 200 240 360 Isothermaltime, min 2 0 0 10 Temperature rate, ° C./min 20 10 12 —

Sample preparation: 50 mg non-polar fraction of wheat lipids wasdissolved in 12 ml heptane:pyridine (2:1) containing 2 mg/ml heptadecaneas internal standard. 500 μl of the solution was transferred to a crimpvial and 100 μl N-methyl-N-trimethylsilyl-trifluoracetamide was added.The mixture was allowed to react for 15 min at 90° C.

Calculation: Response factors for mono-, di- and triglycerides and freefatty acids were determined from reference mixtures of these components.Based on these response factors, the glycerides and the free fatty acidswere calculated in wheat lipids.

8.2. Baking experiments with lipase 3 in Danish toast bread

The effect of adding lipase 3 to a dough for making Danish toast breadwas evaluated. The enzyme was added as a freeze-dried preparation onmaltodextrin together with the other ingredients. The results of thebaking tests are shown in Tables 8.1 to 8.4.

TABLE 8.1 Lipase LUS/kg flour 0 5,000 15,000 25,000 Specific 4.43 4.434.22 4.37 volume of bread Firmness 35 33 32 30 Day 1 Firmness 90 90 8573 Day 7

TABLE 8.2 Lipase LUS/kg flour 0 5,000 15,000 25,000 Average diameter of2.96 2.33 2.47 2.65 the crumb pore, mm Homogeneity of 64.9 73.8 66.067.1 crumb pore, % Porosity, % 85.9 84.7 85.5 85.1 Gluten index, % 4245.5 55 65

TABLE 8.3 Lipase LUS/kg flour 0 5,000 15,000 25,000 Fatty acids, % 0.0900.148 0.218 0.241 Monoglycerides, % 0.017 0.031 0.035 0.039Diglycerides, % 0.020 0.036 0.040 0.045 Triglycerides, % 0.790 0.7140.673 0.622

TABLE 8.4 Lipase LUS/kg flour 0 5,000 15,000 25,000 Monogalactosyl 0.0730.040 0.025 0.018 diglyceride, % Digalactosyl 0.244 0.220 0.182 0.127diglyceride, % Digalactosyl 0.008 0.022 0.044 0.054 monoglyceride, %Phosphatidyl 0.064 0.073 0.055 0.041 choline, % Lysophosphatidyl 0.1640.182 0.171 0.165 choline, %

By the addition of up to about 5,000 LUS/kg flour of the lipase nochange in bread volume was observed, but at a higher dosage of lipase 3there was a tendency to a small but not statistically significantdecrease in volume (Table 8.1).

From the results in Table 8.2 it appears that lipase 3 improved thebread crumb homogeneity and that the average diameter of the crumb poreswas reduced significantly. The gluten index also clearly correlated tothe addition of lipase 3 as an indication of a more firm gluten causedby the modification of the wheat lipid components causing better doughstability and a more homogeneous bread pore structure. However, thesemodifications appeared to be optimal at the addition of 5,000 LUS/kgflour of lipase 3 whereas a higher dosage resulted in a too strongmodification of the wheat gluten.

The results of the GLC and HPLC analyses (Table 8.3) clearlydemonstrated that the triglycerides in the dough were hydrolysed. Butmore interestingly, there was also observed a modification of theglycolipids, monogalactosyl diglyceride and digalactosyl diglyceride.These components were converted to the more polar componentsmonogalactosyl monoglyceride and digalactosyl monoglyceride. Asdigalactosyl monoglyceride is a more surface active component thandigalactosyl diglyceride it is assumed that this component contributedto the observed improved crumb cell structure and homogeneity. It alsoappeared that phospholipids like phosphatidyl choline were only modifiedto a very small extent.

8.3. Baking experiments with lipase 3 in Danish rolls

The effect of adding lipase 3 to a dough for making Danish rolls wasevaluated. The enzyme was added as a freeze-dried preparation onmaltodextrin together with the other ingredients. The results of thebaking tests are shown in Tables 8.5 to 8.7.

TABLE 8.5 Lipase 3 LUS/kg flour 0 10,000 20,000 30,000 Specific volumeof bread 6.86 7.04 6.35 6.36 (45 min fermentation) Specific volume ofbread 8.30 8.59 8.23 8.04 (65 min fermentation) Subjective evaluation of3 5 4 4 crumb homogeneity

TABLE 8.6 Lipase 3 LUS/kg flour 0 10,000 20,000 30,000 Free fatty acids,% 0.060 0.126 0.173 0.211 Monoglycerides, % 0.028 0.050 0.054 0.063Diglycerides, % 0.103 0.095 0.110 0.104 Triglycerides, % 0.705 0.5610.472 0.436

TABLE 8.7 Lipase 3 LUS/kg flour 0 5,000 15,000 25,000 Digalactosyl 0.2040.187 0.154 0.110 diglyceride, % Digalactosyl 0.007 0.026 0.047 0.074monoglyceride, % Phosphatidyl 0.077 0.078 0.077 0.063 choline, %Lysophosphatidyl 0.153 0.161 0.162 0.150 choline, %

It is apparent from the results shown in Table 8.5 that the addition oflipase 3 does not significantly increase the volume of the rolls.Furthermore, lipase 3 was found to improve the homogeneity of the crumb.

The GLC and HPLC analyses of the wheat lipids, as shown in Tables 8.6and 8.7, demonstrated the modification of these lipids.

EXAMPLE 9 Dough improving effect of glycerol oxidase and lipase

The effect of glycerol oxidase and lipase (separately or in combination)on dough strength was studied in a dough prepared according to the AACCMethod 54-10. The dough was subjected to extensiograph measurements(Barbender Extensiograph EXEK/6) also according to AACC Method 54-10with and with out the addition of glycerol oxidase from Aspergillusjaponicus combined with lipase from Aspergillus oryzae (GRIN-DAMYL™ EXEL16, Bakery Enzyme, Danisco Ingredients). The dough with out addition ofenzymes served as a control.

The principle of the above method is that the dough after forming issubjected to a load-extension test after resting at 30° C. for 45, 90and 135 minutes, respectively, using an extensigraph capable ofrecording a load-extension curve (extensigram) which is an indication ofthe doughs resistance to physical deformation when stretched. From thiscurve, the resistance to extension, B (height of curve) and theextensibility, C (total length of curve) can be calculated. The B/Cratio (D) is an indication of the baking strength of the flour dough.The results of the experiment are summarized in Table 9.1 below.

TABLE 9.1 Extensigraph measurements of dough supplemented with glyceroloxidase and lipase Resting Sample time (per kg flour) (min) B-valueC-value D = B/C Control 45 220 192 1.15 500 LUS lipase 45 225 190 1.181000 U glycerol 45 300 195 1.54 oxidase 500 LUS lipase 45 350 198 1.77 +1000 U Glycerol oxidase Control 90 240 196 1.22 500 LUS lipase 90 245195 1.16 1000 U Glycerol 90 330 190 1.74 oxidase 500 LUS lipase 90 380192 1.98 + 1000 U Glycerol oxidase Control 135 260 188 1.38 500 LUSlipase 135 265 190 1.39 1000 U Glycerol 135 380 188 2.02 oxidase 500 LUSlipase 135 410 190 2.15 + 1000 U Glycerol oxidase

When the results from the above experiments are compared with regard tothe differences between the control dough and the glycerol oxidasesupplemented dough it appears that glycerol oxidase clearly has astrengthening effect. The B/C ratio was increased by 34%, 43% and 46%after 45, 90 and 135 minutes of resting time respectively.

The addition of lipase only did not have any effect on the B/C ratio.

However, when supplementing the dough with a combination of glyceroloxidase and lipase, a further increase in the B/C ratio was seen ascompared to bread prepared from dough supplemented with glycerol oxidaseonly. The B/C ratio was increased by 54%, 62% and 56% after 45, 90 and135 minutes respectively. This clearly indicates that the combined useof these two enzymes in the preparation of bread products has anenhancing effect on the baking strength.

EXAMPLE 10 Improvement of the specific volume of bread prepared fromdough supplemented with glycerol oxidase and lipase

The effect of using glycerol oxidase and lipase (separately or incombination) on the specific bread volume and the crumb homogeneity wastested in a baking procedure for Danish rolls with a dough prepared asdescribed in example 8. Glycerol oxidase from Aspergillus japonicus andlipase 3 from Aspergillus tubigensis was added to the dough in differentamounts. Dough without the addition of enzymes served as control. Thefully proofed dough was baked at 220° C. for 18 minutes with 12 secondssteam in a Bago-oven. After cooling the rolls were weighed and thevolume of the rolls were measured by the rape seed displacement method.The specific bread volume was determined as the volume of the bread (ml)divided by the weight of the bread (g). The crumb homogeneity wasevaluated subjectively on a scale from 1 to 7, where 1=courseinhomogeneous and 7=nice homogeneous. The results from this experimentare summarized in Table 10.1 below.

TABLE 10.1 Specific volume and crumb homogeneity in bread supplementedwith lipase and glycerol Sample Specific vol- Crumb homo- (per kg flour)ume (ml/g) geneity Control 5.45 1 1,000 U glycerol oxidase 6.75 2 10,000LUS lipase 5.65 4 10,000 LUS lipase 7.25 7 + 1,000 U glycerol oxidase

As can be seen in the above Tabel 10.1, the use of glycerol oxidase inthe preparing of bread, significantly increased the bread volume (24%)as compared to bread prepared from a similar dough not supplemented withthis enzyme. Addition of glycerol oxidase did not improve the crumbhomogeneity significantly.

The use of lipase in the preparing of bread did not increase thespecific volume of the bread, however a highly increased porehomogeneity was observed.

The combined use of glycerol oxidase and lipase increased the specificvolume of the bread with 33% as compared to bread prepared from asimilar dough not supplemented with any of the two enzymes.

In addition, the crumb homogeneity was highly improved by the combineduse of lipase and glycerol oxidase as compared to the control bread andthe breads prepared from dough supplemented with lipase and glyceroloxidase respectively.

This clearly indicates that the combination of lipase and glyceroloxidase in the preparation of bread has a synergistic effect andsignificantly enhances the shape and appearance of the finished breadproduct.

EXAMPLE 11 Hydrolysis of triglycerides and formation of glycerol indough supplemented with lipase

In order to study the hydrolysis of triglycerides and the formation ofglycerol in a proofed dough supplemented with lipase, a dough for Danishrolls was prepared in the same manner as described in example 8.Different amounts of lipase (GRINDAMYL™ EXEL 16) was added to the dough,and the total lipid from the fully proofed dough was extracted andanalyzed by gas chromatography as described above.

TABLE 11.1 Triglycerides and glycerol in a dough as a func- tion oflipase addition Lipase addition (GRINDAMYL ™ EXEL 16) GlycerolTriglycerides (LUS per kg flour) (%) (%) 0 2.2 7.88 500 2.2 6.22 1,2502.4 5.99 2,500 2.8 5.37 3,750 2.9 5.47 5,000 3.0 5.55 7,500 3.1 5.0310,000 3.0 4.39

From the above experiment it is clear that the addition of lipase to adough has a hydrolyzing effect on the triglycerides present in thedough, which is seen as a decrease in the triglyceride content asfunction of the increased lipase addition. The resulting level ofglycerol increases as a function of the lipase addition. These resultssuggests, that the improvement of the B/C ratio and the specific breadvolume in bread prepared from dough supplemented with both glyceroloxidase and lipase, as was seen in example 9 and 10, could be due tothat lipase addition to a dough is generating glycerol which further canact as substrate for glycerol oxidase.

18 1 25 PRT Aspergillus tubingensis UNSURE (22)..(22) UNSURE 1 Ser ValSer Thr Ser Thr Leu Asp Glu Leu Gln Leu Phe Ala Gln Trp 1 5 10 15 SerAla Ala Ala Tyr Xaa Ser Asn Asn 20 25 2 7 PRT Aspergillus tubingensis 2Val His Thr Gly Phe Trp Lys 1 5 3 14 PRT Aspergillus tubingensis 3 AlaTrp Glu Ser Ala Ala Asp Glu Leu Thr Ser Lys Ile Lys 1 5 10 4 20 DNAArtificial PCR primer 4 ttccaraanc cngtrtgnac 20 5 18 DNA Artificial PCRprimer 5 carytnttyg cncartgg 18 6 17 DNA Artificial PCR primer 6gcvgchswyt cccavgc 17 7 317 DNA Aspergillus tubingensis 7 cagttgttcgcgcaatggtc tgccgcagct tattgctcga ataatatcga ctcgaaagav 60 tccaacttgacatgcacggc caacgcctgt ccatcagtcg aggaggccag taccacgatg 120 ctgctggagttcgacctgta tgtcactcag atcgcagaca tagagcacag ctaattgaac 180 aggacgaacgacttttggag gcacagccgg tttcctggcc gcggacaaca ccaacaagcg 240 gctcgtggtcgccttccggg gaagcagcac gattgagaac tggattgcta atcytgactt 300 catcctggragataacg 317 8 1045 DNA Aspergillus tubingensis 8 atgttctctg gacggtttggagtgcttttg acagcgcttg ctgcgctggg tgctgccgcg 60 ccggcaccgc ttgctgtgcggagtaggtgt gcccgatgtg agatggttgg atagcactga 120 tgaagggtga ataggtgtctcgacttccac gttggatgag ttgcaattgt tcgcgcaatg 180 gtctgccgca gcttattgctcgaataatat cgactcgaaa gactccaact tgacatgcac 240 ggccaacgcc tgtccatcagtcgaggaggc cagtaccacg atgctgctgg agttcgacct 300 gtatgtcact cagatcgcagacatagagca cagctaattt gaacaggacg aacgactttg 360 gaggcacagc cggtttcctggccgcggaca acaccaacaa gcggctcgtg gtcgccttcc 420 ggggaagcag cacgattgagaactggattg ctaatcttga cttcatcctg gaagataacg 480 acgacctctg caccggctgcaaggtccata ctggtttctg gaaggcatgg gagtccgctg 540 ccgacgaact gacgagcaagatcaagtctg cgatgagcac gtattcgggc tataccctat 600 acttcaccgg gcacagtttgggcggcgcat tggctacgct gggagcgaca gttctgcgaa 660 atgacggata tagcgttgagctggtgagtc cttcacaaag gtgatggagc gacaatcggg 720 aacagacagt caatagtacacctatggatg tcctcgaatc ggaaactatg cgctggctga 780 gcatatcacc agtcagggatctggggccaa cttccgtgtt acacacttga acgacatcgt 840 cccccgggtg ccacccatggactttggatt cagtcagcca agtccggaat actggatcac 900 cagtggcaat ggagccagtgtcacggcgtc ggatatcgaa gtcatcgagg gaatcaattc 960 aacggcggga aatgcaggcgaagcaacggt gagcgttgtg gctcacttgt ggtacttttt 1020 tgcgatttcc gagtgcctgctataa 1045 9 297 PRT Aspergillus tubingensis 9 Met Phe Ser Gly Arg PheGly Val Leu Leu Thr Ala Leu Ala Ala Leu 1 5 10 15 Gly Ala Ala Ala ProAla Pro Leu Ala Val Arg Ser Val Ser Thr Ser 20 25 30 Thr Leu Asp Glu LeuGln Leu Phe Ala Gln Trp Ser Ala Ala Ala Tyr 35 40 45 Cys Ser Asn Asn IleAsp Ser Lys Asp Ser Asn Leu Thr Cys Thr Ala 50 55 60 Asn Ala Cys Pro SerVal Glu Glu Ala Ser Thr Thr Met Leu Leu Glu 65 70 75 80 Phe Asp Leu ThrAsn Asp Phe Gly Gly Thr Ala Gly Phe Leu Ala Ala 85 90 95 Asp Asn Thr AsnLys Arg Leu Val Val Ala Phe Arg Gly Ser Ser Thr 100 105 110 Ile Glu AsnTrp Ile Ala Asn Leu Asp Phe Ile Leu Glu Asp Asn Asp 115 120 125 Asp LeuCys Thr Gly Cys Lys Val His Thr Gly Phe Trp Lys Ala Trp 130 135 140 GluSer Ala Ala Asp Glu Leu Thr Ser Lys Ile Lys Ser Ala Met Ser 145 150 155160 Thr Tyr Ser Gly Tyr Thr Leu Tyr Phe Thr Gly His Ser Leu Gly Gly 165170 175 Ala Leu Ala Thr Leu Gly Ala Thr Val Leu Arg Asn Asp Gly Tyr Ser180 185 190 Val Glu Leu Tyr Thr Tyr Gly Cys Pro Arg Ile Gly Asn Tyr AlaLeu 195 200 205 Ala Glu His Ile Thr Ser Gln Gly Ser Gly Ala Asn Phe ArgVal Thr 210 215 220 His Leu Asn Asp Ile Val Pro Arg Val Pro Pro Met AspPhe Gly Phe 225 230 235 240 Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr SerGly Asn Gly Ala Ser 245 250 255 Val Thr Ala Ser Asp Ile Glu Val Ile GluGly Ile Asn Ser Thr Ala 260 265 270 Gly Asn Ala Gly Glu Ala Thr Val SerVal Val Ala His Leu Trp Tyr 275 280 285 Phe Phe Ala Ile Ser Glu Cys LeuLeu 290 295 10 392 PRT Rhizopus delamar 10 Met Val Ser Phe Ile Ser IleSer Gln Gly Val Ser Leu Cys Leu Leu 1 5 10 15 Val Ser Ser Met Met LeuGly Ser Ser Ala Val Pro Val Ser Gly Lys 20 25 30 Ser Gly Ser Ser Asn ThrAla Val Ser Ala Ser Asp Asn Ala Ala Leu 35 40 45 Pro Pro Leu Ile Ser SerArg Cys Ala Pro Pro Ser Asn Lys Gly Ser 50 55 60 Lys Ser Asp Leu Gln AlaGlu Pro Tyr Asn Met Gln Lys Asn Thr Glu 65 70 75 80 Trp Tyr Glu Ser HisGly Gly Asn Leu Thr Ser Ile Gly Lys Arg Asp 85 90 95 Asp Asn Leu Val GlyGly Met Thr Leu Asp Leu Pro Ser Asp Ala Pro 100 105 110 Pro Ile Ser LeuSer Ser Ser Thr Asn Ser Ala Ser Asp Gly Gly Lys 115 120 125 Val Val AlaAla Thr Thr Ala Gln Ile Gln Glu Phe Thr Lys Tyr Ala 130 135 140 Gly IleAla Ala Thr Ala Tyr Cys Arg Ser Val Val Pro Gly Asn Lys 145 150 155 160Trp Asp Cys Val Gln Cys Gln Lys Trp Val Pro Asp Gly Lys Ile Ile 165 170175 Thr Thr Phe Thr Ser Leu Leu Ser Asp Thr Asn Gly Tyr Val Leu Arg 180185 190 Ser Asp Lys Gln Lys Thr Ile Tyr Leu Val Phe Arg Gly Thr Asn Ser195 200 205 Phe Arg Ser Ala Ile Thr Asp Ile Val Phe Asn Phe Ser Asp TyrLys 210 215 220 Pro Val Lys Gly Ala Lys Val His Ala Gly Phe Leu Ser SerTyr Glu 225 230 235 240 Gln Val Val Asn Asp Tyr Phe Pro Val Val Gln GluGln Leu Thr Ala 245 250 255 His Pro Thr Tyr Lys Val Ile Val Thr Gly HisSer Leu Gly Gly Ala 260 265 270 Gln Ala Leu Leu Ala Gly Met Asp Leu TyrGln Arg Glu Pro Arg Leu 275 280 285 Ser Pro Lys Asn Leu Ser Ile Phe ThrVal Gly Gly Pro Arg Val Gly 290 295 300 Asn Pro Thr Phe Ala Tyr Tyr ValGlu Ser Thr Gly Ile Pro Phe Gln 305 310 315 320 Arg Thr Val His Lys ArgAsp Ile Val Pro His Val Pro Pro Gln Ser 325 330 335 Phe Gly Phe Leu HisPro Gly Val Glu Ser Trp Ile Lys Ser Gly Thr 340 345 350 Ser Asn Val GlnIle Cys Thr Ser Glu Ile Glu Thr Lys Asp Cys Ser 355 360 365 Asn Ser IleVal Pro Phe Thr Ser Ile Leu Asp His Leu Ser Tyr Phe 370 375 380 Asp IleAsn Glu Gly Ser Cys Leu 385 390 11 363 PRT Rhizomucor miehei 11 Met ValLeu Lys Gln Arg Ala Asn Tyr Leu Gly Phe Leu Ile Val Phe 1 5 10 15 PheThr Ala Phe Leu Val Glu Ala Val Pro Ile Lys Arg Gln Ser Asn 20 25 30 SerThr Val Asp Ser Leu Pro Pro Leu Ile Pro Ser Arg Thr Ser Ala 35 40 45 ProSer Ser Ser Pro Ser Thr Thr Asp Pro Glu Ala Pro Ala Met Ser 50 55 60 ArgAsn Gly Pro Leu Pro Ser Asp Val Glu Thr Lys Tyr Gly Met Ala 65 70 75 80Leu Asn Ala Thr Ser Tyr Pro Asp Ser Val Val Gln Ala Met Ser Ile 85 90 95Asp Gly Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu Leu Thr 100 105110 Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val Ile Pro 115120 125 Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp Leu Lys130 135 140 Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala MetVal 145 150 155 160 Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val PheArg Gly Ser 165 170 175 Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr PheVal Pro Val Ser 180 185 190 Tyr Pro Pro Val Ser Gly Thr Lys Val His LysGly Phe Leu Asp Ser 195 200 205 Tyr Gly Glu Val Gln Asn Glu Leu Val AlaThr Val Leu Asp Gln Phe 210 215 220 Lys Gln Tyr Pro Ser Tyr Lys Val AlaVal Thr Gly His Ser Leu Gly 225 230 235 240 Gly Ala Thr Ala Leu Leu CysAla Leu Asp Leu Tyr Gln Arg Glu Glu 245 250 255 Gly Leu Ser Ser Ser AsnLeu Phe Leu Tyr Thr Gln Gly Gln Pro Arg 260 265 270 Val Gly Asp Pro AlaPhe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro 275 280 285 Tyr Arg Arg ThrVal Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro 290 295 300 Ala Ala PheGly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile Thr Asp 305 310 315 320 AsnSer Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu Thr Ser 325 330 335Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu 340 345350 Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr 355 360 12 305 PRTPenicillium camemberti 12 Met Arg Leu Ser Phe Phe Thr Ala Leu Ser AlaVal Ala Ser Leu Gly 1 5 10 15 Tyr Ala Leu Pro Gly Lys Leu Gln Ser ArgAsp Val Ser Thr Ser Glu 20 25 30 Leu Asp Gln Phe Glu Phe Trp Val Gln TyrAla Ala Ala Ser Tyr Tyr 35 40 45 Glu Ala Asp Tyr Thr Ala Gln Val Gly AspLys Leu Ser Cys Ser Lys 50 55 60 Gly Asn Cys Pro Glu Val Glu Ala Thr GlyAla Thr Val Ser Tyr Asp 65 70 75 80 Phe Ser Asp Ser Thr Ile Thr Asp ThrAla Gly Tyr Ile Ala Val Asp 85 90 95 His Thr Asn Ser Ala Val Val Leu AlaPhe Arg Gly Ser Tyr Ser Val 100 105 110 Arg Asn Trp Val Ala Asp Ala ThrPhe Val His Thr Asn Pro Gly Leu 115 120 125 Cys Asp Gly Cys Leu Ala GluLeu Gly Phe Trp Ser Ser Trp Lys Leu 130 135 140 Val Arg Asp Asp Ile IleLys Glu Leu Lys Glu Val Val Ala Gln Asn 145 150 155 160 Pro Asn Tyr GluLeu Val Val Val Gly His Ser Leu Gly Ala Ala Val 165 170 175 Ala Thr LeuAla Ala Thr Asp Leu Arg Gly Lys Gly Tyr Pro Ser Ala 180 185 190 Lys LeuTyr Ala Tyr Ala Ser Pro Arg Val Gly Asn Ala Ala Leu Ala 195 200 205 LysTyr Ile Thr Ala Gln Gly Asn Asn Phe Arg Phe Thr His Thr Asn 210 215 220Asp Pro Val Pro Lys Leu Pro Leu Leu Ser Met Gly Tyr Val His Val 225 230235 240 Ser Pro Glu Tyr Trp Ile Thr Ser Pro Asn Asn Ala Thr Val Ser Thr245 250 255 Ser Asp Ile Lys Val Ile Asp Gly Asp Val Ser Phe Asp Gly AsnThr 260 265 270 Gly Thr Gly Leu Pro Leu Leu Thr Asp Phe Glu Ala His IleTrp Tyr 275 280 285 Phe Val Gln Val Asp Ala Gly Lys Gly Pro Gly Leu ProPhe Lys Arg 290 295 300 Val 305 13 334 DNA Aspergillus tubingensisUnsure (10)..(10) Unsure 13 tacccggggn tccgatt cag ttg ttc gcg caa tggtct gcc gca gct tat 50 Gln Leu Phe Ala Gln Trp Ser Ala Ala Ala Tyr 1 510 tgc tcg aat aat atc gac tcg aaa gav tcc aac ttg aca tgc acg gcc 98Cys Ser Asn Asn Ile Asp Ser Lys Xaa Ser Asn Leu Thr Cys Thr Ala 15 20 25aac gcc tgt cca tca gtc gag gag gcc agt acc acg atg ctg ctg gag 146 AsnAla Cys Pro Ser Val Glu Glu Ala Ser Thr Thr Met Leu Leu Glu 30 35 40 ttcgac ctg tat gtc act cag atc gca gac ata gag cac agc taa ttg 194 Phe AspLeu Tyr Val Thr Gln Ile Ala Asp Ile Glu His Ser Leu 45 50 55 aac agg acgaac gac ttt tgg agg cac agc cgg ttt cct ggc cgc gga 242 Asn Arg Thr AsnAsp Phe Trp Arg His Ser Arg Phe Pro Gly Arg Gly 60 65 70 caa cac caa caagcg gct cgt ggt cgc ctt ccg ggg aag cag cac gat 290 Gln His Gln Gln AlaAla Arg Gly Arg Leu Pro Gly Lys Gln His Asp 75 80 85 90 tga gaa ctg gattgc taa tcy tga ctt cat cct ggr aga taacg 334 Glu Leu Asp Cys Xaa LeuHis Pro Xaa Arg 95 100 14 57 PRT Aspergillus tubingensis UNSURE(20)..(20) UNSURE 14 Gln Leu Phe Ala Gln Trp Ser Ala Ala Ala Tyr Cys SerAsn Asn Ile 1 5 10 15 Asp Ser Lys Xaa Ser Asn Leu Thr Cys Thr Ala AsnAla Cys Pro Ser 20 25 30 Val Glu Glu Ala Ser Thr Thr Met Leu Leu Glu PheAsp Leu Tyr Val 35 40 45 Thr Gln Ile Ala Asp Ile Glu His Ser 50 55 15 33PRT Aspergillus tubingensis 15 Leu Asn Arg Thr Asn Asp Phe Trp Arg HisSer Arg Phe Pro Gly Arg 1 5 10 15 Gly Gln His Gln Gln Ala Ala Arg GlyArg Leu Pro Gly Lys Gln His 20 25 30 Asp 16 4 PRT Aspergillustubingensis 16 Glu Leu Asp Cys 1 17 5 PRT Aspergillus tubingensis UNSURE(4)..(4) UNSURE 17 Leu His Pro Xaa Arg 1 5 18 1833 DNA Aspergillustubingensis Unsure (3)..(3) Unsure 18 ccndttaatc ccccaccggg gttcccgctcccggatggag atggggccaa aactggcaac 60 ccccagttgc gcaacggaac aaccgccgacccggaacaaa ggatgcggat gaggagatac 120 ggtgcctgat tgcatggctg gcttcatctgctatcgtgac agtgctcttt gggtgaatat 180 tgttgtctga cttaccccgc ttcttgctttttcccccctg aggccctgat ggggaatcgc 240 ggtgggtaat atgatatggg tataaaagggagatcggagg tgcagttgga ttgaggcagt 300 gtgtgtgtgt gcattgcaga agcccgttggtcgcaaggtt ttggtcgcct cgattgtttg 360 tataccgcaa g atg ttc tct gga cggttt gga gtg ctt ttg aca gcg ctt 410 Met Phe Ser Gly Arg Phe Gly Val LeuLeu Thr Ala Leu 1 5 10 gct gcg ctg ggt gct gcc gcg ccg gca ccg ctt gctgtg cgg a 453 Ala Ala Leu Gly Ala Ala Ala Pro Ala Pro Leu Ala Val Arg 1520 25 gtaggtgtgc ccgatgtgag atggttggat agcactgatg aagggtgaat ag gt gtc510 Ser Val tcg act tcc acg ttg gat gag ttg caa ttg ttc gcg caa tgg tctgcc 558 Ser Thr Ser Thr Leu Asp Glu Leu Gln Leu Phe Ala Gln Trp Ser Ala30 35 40 45 gca gct tat tgc tcg aat aat atc gac tcg aaa gac tcc aac ttgaca 606 Ala Ala Tyr Cys Ser Asn Asn Ile Asp Ser Lys Asp Ser Asn Leu Thr50 55 60 tgc acg gcc aac gcc tgt cca tca gtc gag gag gcc agt acc acg atg654 Cys Thr Ala Asn Ala Cys Pro Ser Val Glu Glu Ala Ser Thr Thr Met 6570 75 ctg ctg gag ttc gac ctg tatgtcactc agatcgcaga catagagcac 702 LeuLeu Glu Phe Asp Leu 80 agctaatttg aacagg acg aac gac ttt gga ggc aca gccggt ttc ctg gcc 754 Thr Asn Asp Phe Gly Gly Thr Ala Gly Phe Leu Ala 8590 95 gcg gac aac acc aac aag cgg ctc gtg gtc gcc ttc cgg gga agc agc802 Ala Asp Asn Thr Asn Lys Arg Leu Val Val Ala Phe Arg Gly Ser Ser 100105 110 acg att gag aac tgg att gct aat ctt gac ttc atc ctg gaa gat aac850 Thr Ile Glu Asn Trp Ile Ala Asn Leu Asp Phe Ile Leu Glu Asp Asn 115120 125 gac gac ctc tgc acc ggc tgc aag gtc cat act ggt ttc tgg aag gca898 Asp Asp Leu Cys Thr Gly Cys Lys Val His Thr Gly Phe Trp Lys Ala 130135 140 tgg gag tcc gct gcc gac gaa ctg acg agc aag atc aag tct gcg atg946 Trp Glu Ser Ala Ala Asp Glu Leu Thr Ser Lys Ile Lys Ser Ala Met 145150 155 agc acg tat tcg ggc tat acc cta tac ttc acc ggg cac agt ttg ggc994 Ser Thr Tyr Ser Gly Tyr Thr Leu Tyr Phe Thr Gly His Ser Leu Gly 160165 170 175 ggc gca ttg gct acg ctg gga gcg aca gtt ctg cga aat gac ggatat 1042 Gly Ala Leu Ala Thr Leu Gly Ala Thr Val Leu Arg Asn Asp Gly Tyr180 185 190 agc gtt gag ctg gtgagtcctt cacaaaggtg atggagcgac aatcgggaac1094 Ser Val Glu Leu 195 agacagtcaa tag tac acc tat gga tgt cct cga atcgga aac tat gcg 1143 Tyr Thr Tyr Gly Cys Pro Arg Ile Gly Asn Tyr Ala 200205 ctg gct gag cat atc acc agt cag gga tct ggg gcc aac ttc cgt gtt 1191Leu Ala Glu His Ile Thr Ser Gln Gly Ser Gly Ala Asn Phe Arg Val 210 215220 aca cac ttg aac gac atc gtc ccc cgg gtg cca ccc atg gac ttt gga 1239Thr His Leu Asn Asp Ile Val Pro Arg Val Pro Pro Met Asp Phe Gly 225 230235 ttc agt cag cca agt ccg gaa tac tgg atc acc agt ggc aat gga gcc 1287Phe Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Ser Gly Asn Gly Ala 240 245250 255 agt gtc acg gcg tcg gat atc gaa gtc atc gag gga atc aat tca acg1335 Ser Val Thr Ala Ser Asp Ile Glu Val Ile Glu Gly Ile Asn Ser Thr 260265 270 gcg gga aat gca ggc gaa gca acg gtg agc gtt gtg gct cac ttg tgg1383 Ala Gly Asn Ala Gly Glu Ala Thr Val Ser Val Val Ala His Leu Trp 275280 285 tac ttt ttt gcg att tcc gag tgc ctg cta taactagacc gactgtcaga1433 Tyr Phe Phe Ala Ile Ser Glu Cys Leu Leu 290 295 ttagtggacgggagaagtgt acataagtaa ttagtatata atcagagcaa cccagtggtg 1493 gtgatggtggtgaaagaaga aacacattga gttcccatta cgkagcagwt aaagcacktk 1553 kggaggcgctggttcctcca cttggcagtt ggcggccatc aatcatcttt cctctcctta 1613 ctttcgtccaccacaactcc catcctgcca gctgtcgcat ccccgggttg caacaactat 1673 cgcctccggggcctccgtgg ttctcctata ttattccatc cgacggccga cgtttcaccc 1733 tcaacctgcgccgccgcaaa atctccccga gtcggtcaac tccctcgaac cgccgcccgc 1793 atcgacctcacgaccccgac cgtctgygat ygtccaaccg 1833

What is claimed is:
 1. A method of altering the rheological propertiesof a flour dough and the quality of the finished product made from thedough, comprising adding to the dough a glycerol oxidase, which does notrequire a co-factor to oxidize glycerol, and a lipase, wherein thecombined effect of the glycerol oxidase and the lipase on saidrheological properties is synergistic.
 2. A method according to claim 1wherein the amount of the glycerol oxidase added is in the range of 10to 10,000 units per kg flour.
 3. A method according to claim 2 whereinthe glycerol oxidase is derived from an organism selected from the groupconsisting of a bacterial species, a fungal species, a yeast species, ananimal cell and a plant cell.
 4. A method according to claim 3 whereinthe fungal species comprises an Aspergillus species, a Neurosporaspecies or a Penicillium species.
 5. A method according to claim 4wherein the Aspergillus species comprises A. japonicus, A. oryzae, A.parasiticus, or A. flavus.
 6. A method according to claim 4 wherein theNeurospora species comprises N. crassa, N. sitophila, or N. tetrasperma.7. A method according to claim 4 wherein the Penicillium speciescomprises P. nigricans, P. funiculosum or P. janthinellum.
 8. A methodaccording to claim 1 wherein the amount of the lipase added is in therange of 10 to 100,000 LUS per kg of flour.
 9. A method according toclaim 1 wherein the lipase is derived from an organism comprising abacterial species, a fungal species, a yeast species, an animal cell ora plant cell.
 10. A method according to claim 9 wherein the lipase isderived from an Aspergillus species.
 11. A method according to claim 10wherein the Aspergillus species comprises A. tubigensis, A. oryzae or A.niger.
 12. A method according to claim 1 wherein the lipase comprises atleast one of the following amino acid sequences: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:9.
 13. A method according to claim 1wherein the alteration of the rheological properties includes anincrease in the resistance to extension of the dough, in terms of theratio between resistance to extension (height of curve, B) and theextensibility (length of curve, C), expressed as the B/C ratio, asmeasured by the AACC method 54-10, relative to that of an otherwisesimilar dough not containing glycerol oxidase and lipase.
 14. A methodaccording to claim 13 wherein the resistance to extension of the doughis increased by at least 10%.
 15. A method according to claim 13 whereinthe resistance to extension of the dough is increased by at least 25%.16. A method according to claim 1 wherein the alteration of the qualityof the finished product made from the dough includes an increase in thepore homogeneity of the crumb of the bread made from the dough by atleast 5%, relative to a bread which is made from a bread dough withoutaddition of the glycerol oxidase and lipase.
 17. A method according toclaim 1 wherein the alteration of the rheological properties includes anincrease in the specific volume of the finished product relative to anotherwise similar product prepared from a dough not containing lipaseand glycerol oxidase.
 18. A method according to claim 17 wherein thespecific volume is increased by at least 10%.
 19. A method according toclaim 17 wherein the specific volume is increased by at least 30%.
 20. Amethod according to claim 1 wherein the finished product comprises atleast one of a bread product, a noodle product or an alimentary pasteproduct.
 21. A method according to claim 1 wherein at least one furtherenzyme is added to the dough ingredients, dough additives or the dough.22. A method according to claim 21 wherein the further enzyme comprisesa cellulase, a hemicellulase, a starch degrading enzyme, anoxidoreductase, or a protease.
 23. A flour dough altering compositionwhich is a pre-mixture containing all of the dry ingredients andadditives for a flour dough, and further comprising a glycerol oxidase,which does not require a co-factor to oxidize glycerol, and a lipase andat least one further dough additive comprising at least one of amonoglyceride, a diacetyl tartaric acid ester of mono- or diglyceridesof fatty acids, a starch degrading enzyme, a cellulose or hemicellulosedegrading enzyme, a protease, a non-specific oxidizing agent, aflavoring agent, a lactic acid bacterial culture, a vitamin, a mineral,an alginate, a carrageenan, a pectin, a vegetable gum, or a dietaryfiber substance, which composition, when it is added to a flour dough,results in a synergistic effect of the glycerol oxidase and the lipaseon the rheological properties of the flour dough and the quality of thefinished product made from the flour dough.
 24. A flour dough alteringcomposition according to claim 23, wherein the synergistic effect on therheological properties of the flour dough and the quality of thefinished product includes: i) increased resistance to extension of thedough, ii) increased pore homogeneity of the crumb of the bread madefrom the dough or, iii) increased specific volume of the finishedproduct.
 25. A composition according to claim 23 wherein the furtherdough additive additionally comprises a substrate for glycerol oxidase,an emulsifying agent or a hydrocolloid.
 26. A composition according toclaim 25 wherein the hydrocolloid comprises an alginate, a carrageenan,a pectin or a vegetable gum.
 27. A method of preparing a finishedproduct, made from a flour dough, which comprises at least one of abaked product, a noodle product or an alimentary paste product,comprising adding to the flour dough a pre-mixture which includes about10 to 10,000 units per kg flour of a glycerol oxidase, which does notrequire a co-factor to oxidize glycerol, and about 10 to 100,000 LUS perkg flour of lipase, which pre-mixture, when it is added to the flourdough, results in a synergistic effect of the glycerol oxidase and thelipase on the rheological properties of the flour dough and the qualityof the finished product made from the flour dough.
 28. A methodaccording to claim 13 wherein the resistance to extension of the doughis increased by at least 50%.
 29. A method according to claim 13 whereinthe resistance to extension of the dough is increased by at least 75%.30. The composition of claim 23 wherein the amount of glycerol oxidaseis in the range of 10 to 5,000 units per kg flour.
 31. The compositionof claim 23 wherein the amount of lipase is in the range of 10 to 20,000LUS per kg flour.